Novel compound useful for the treatment of degenerative and inflammatory diseases

ABSTRACT

A compound is disclosed that has a formula represented by the following: 
     
       
         
         
             
             
         
       
     
     This compound may be prepared as a pharmaceutical composition, and may be used for the prevention and treatment of a variety of conditions in mammals including humans, including by way of non-limiting example, inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6.

RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. §119 of U.S.Provisional Application No. 61/220,685, filed Jun. 26, 2009, andProvisional Application No. 61/298,188, filed Jan. 25, 2010. Thecontents of both applications are hereby incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a compound that is an inhibitor of JAK,a family of tyrosine kinases that are involved in inflammatoryconditions, autoimmune diseases, proliferative diseases, transplantationrejection, diseases involving impairment of cartilage turnover,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6. The present invention also provides methods forthe production of this compound, pharmaceutical compositions comprisingthis compound, and methods for the prevention and/or treatment ofinflammatory conditions, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6 by administering a compound of the invention.

Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transducecytokine signaling from membrane receptors to STAT transcriptionfactors. Four JAK family members are described, JAK1, JAK2, JAK3 andTYK2. Upon binding of the cytokine to its receptor, JAK family membersauto- and/or transphosphorylate each other, followed by phosphorylationof STATs that then migrate to the nucleus to modulate transcription.JAK-STAT intracellular signal transduction serves the interferons, mostinterleukins, as well as a variety of cytokines and endocrine factorssuch as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF, and PRL (Vainchenker W. etal. (2008).

The combination of genetic models and small molecule JAK inhibitorresearch revealed the therapeutic potential of several JAKs. JAK3 isvalidated by mouse and human genetics as an immune-suppression target(O'Shea J. et al. (2004)). JAK3 inhibitors were successfully taken intoclinical development, initially for organ transplant rejection but lateralso in other immuno-inflammatory indications such as rheumathoidarthritis (RA), psoriasis and Crohn's disease(http://clinicaltrials.gov/).

TYK2 is a potential target for immuno-inflammatory diseases, beingvalidated by human genetics and mouse knock-out studies (Levy D. andLoomis C. (2007)).

JAK1 is a novel target in the immuno-inflammatory disease area. JAK1heterodimerizes with the other JAKs to transduce cytokine-drivenpro-inflammatory signaling. Therefore, inhibition of JAK1 and/or otherJAKs is expected to be of therapeutic benefit for a range ofinflammatory conditions as well as for other diseases driven byJAK-mediated signal transduction.

BACKGROUND OF THE INVENTION

The degeneration of cartilage is the hallmark of various diseases, amongwhich rheumatoid arthritis and osteoarthritis are the most prominent.Rheumatoid arthritis (RA) is a chronic joint degenerative disease,characterized by inflammation and destruction of the joint structures.When the disease is unchecked, it leads to substantial disability andpain due to loss of joint functionality and even premature death. Theaim of an RA therapy, therefore, is not only to slow down the diseasebut to attain remission in order to stop the joint destruction. Besidesthe severity of the disease outcome, the high prevalence of RA (˜0.8% ofthe adults are affected worldwide) means a high socio-economic impact.(For reviews on RA, we refer to Smolen and Steiner (2003); Lee andWeinblatt (2001); Choy and Panayi (2001); O'Dell (2004) and Firestein(2003)).

Osteoarthritis (also referred to as OA, or wear-and-tear arthritis) isthe most common form of arthritis and is characterized by loss ofarticular cartilage, often associated with hypertrophy of the bone andpain. For an extensive review on osteoarthritis, we refer to Wieland etal., (2005).

Osteoarthritis is difficult to treat. At present, no cure is availableand treatment focuses on relieving pain and preventing the affectedjoint from becoming deformed. Common treatments include the use ofnon-steroidal anti-inflammatory drugs (NSAIDs). Although dietarysupplements such as chondroitin and glucosamine sulphate have beenadvocated as safe and effective options for the treatment ofosteoarthritis, a recent clinical trial revealed that both treatmentsdid not reduce pain associated to osteoarthritis. (Clegg et al., 2006).Taken together, no disease modifying osteoarthritic drugs are available.

Stimulation of the anabolic processes, blocking catabolic processes, ora combination of these two, may result in stabilization of thecartilage, and perhaps even reversion of the damage, and thereforeprevent further progression of the disease. Various triggers maystimulate anabolic stimulation of chondrocytes. Insulin-like growthfactor-I (IGF-I) is the predominant anabolic growth factor in synovialfluid and stimulates the synthesis of both proteoglycans and collagen.It has also been shown that members of the bone morphogenetic protein(BMP) family, notably BMP2, BMP4, BMP6, and BMP7, and members of thehuman transforming growth factor-β (TGF-β) family can induce chondrocyteanabolic stimulation (Chubinskaya and Kuettner, 2003). A compound hasrecently been identified that induces anabolic stimulation ofchondrocytes (U.S. Pat. No. 6,500,854; EP 1 391 211). However, most ofthese compounds show severe side effects and, consequently, there is astrong need for compounds that stimulate chondrocyte differentiationwithout these side effects.

Vandeghinste et al. (WO 2005/124342) discovered JAK1 as a target whoseinhibition might have therapeutic relevance for several diseasesincluding OA. JAK1 belongs to the Janus kinase (JAK) family ofcytoplasmic tyrosine kinases, involved in cytokine receptor-mediatedintracellular signal transduction. The JAK family consists of 4 members:JAK1, JAK2, JAK3 and TYK2. JAKs are recruited to cytokine receptors,upon binding of the cytokine, followed by heterodimerization of thecytokine receptor and a shared receptor subunit (common gamma-c chain,gp130). JAKs are then activated by auto- and/or transphosphorylation byanother JAK, resulting in phosphorylation of the receptors andrecruitment and phosphorylation of members of the signal transducer andactivator of transcription (STATs). Phosphorylated STATs dimerize andtranslocate to the nucleus where they bind to enhancer regions ofcytokine-responsive genes. Knockout of the JAK1 gene in micedemonstrated that JAK1 plays essential and nonredundant roles duringdevelopment: JAK1−/− mice died within 24 h after birth and lymphocytedevelopment was severely impaired. Moreover, JAK1−/− cells were not, orless, reactive to cytokines that use class TT cytokine receptors,cytokine receptors that use the gamma-c subunit for signaling and thefamily of cytokine receptors that use the gp130 subunit for signaling(Rodig et al., 1998).

Various groups have implicated JAK-STAT signaling in chondrocytebiology. Li et al. (2001) showed that Oncostatin M induces MMP and TIMP3gene expression in primary chondrocytes by activation of JAK/STAT andMAPK signaling pathways. Osaki et al. (2003) showed thatinterferon-gamma mediated inhibition of collagen II in chondrocytesinvolves JAK-STAT signaling. IL1-beta induces cartilage catabolism byreducing the expression of matrix components, and by inducing theexpression of collagenases and inducible nitric oxide synthase (NOS2),which mediates the production of nitric oxide (NO). Otero et al., (2005)showed that leptin and IL1-beta synergistically induced NO production orexpression of NOS2 mRNA in chondrocytes, and that that was blocked by aJAK inhibitor. Legendre et al. (2003) showed that IL6/IL6Receptorinduced downregulation of cartilage-specific matrix genes collagen II,aggrecan core and link protein in bovine articular chondrocytes, andthat this was mediated by JAK/STAT signaling. Therefore, theseobservations suggest a role for JAK kinase activity in cartilagehomeostasis and therapeutic opportunities for JAK kinase inhibitors.

JAK family members have been implicated in additional conditionsincluding myeloproliferative disorders (O'Sullivan et al, 2007, MolImmunol 44(10):2497-506), where mutations in JAK2 have been identified.This indicates that inhibitors of JAK in particular JAK2 may also be ofuse in the treatment of myeloproliferative disorders. Additionally, theJAK family, in particular JAK1, JAK2 and JAK3, has been linked tocancers, in particular leukaemias e.g. acute myeloid leukaemia(O'Sullivan et al, 2007, Mol. Immunol. 44(10):2497-506; Xiang et al.,2008, “Identification of somatic JAK1 mutations in patients with acutemyeloid leukemia” Blood First Edition Paper, prepublished online Dec.26, 2007; DOI 10.1182/blood-2007-05-090308) and acute lymphoblasticleukemia (Mullighan et al, 2009) or solid tumours e.g. uterineleiomyosarcoma (Constantinescu et al., 2007, Trends in BiochemicalSciences 33(3): 122-131), prostate cancer (Tam et al., 2007, BritishJournal of Cancer, 97, 378-383). These results indicate that inhibitorsof JAK, in particular of JAK1 and/or JAK2, may also have utility in thetreatment of cancers (leukaemias and solid tumours e.g. uterineleiomyosarcoma, prostate cancer).

In addition, Castleman's disease, multiple myeloma, mesangialproliferative glomerulonephritis, psoriasis, and Kaposi's sarcoma arelikely due to hypersecretion of the cytokine IL-6, whose biologicaleffects are mediated by intracellular JAK-STAT signaling (Tetsuji Naka,Norihiro Nishimoto and Tadamitsu Kishimoto, Arthritis Res 2002, 4 (suppl3):S233-S242). This result shows that inhibitor of JAK, may also findutility in the treatment of said diseases.

A link with autoimmune diseases has been established for JAK3 and Tyk2.Mutations in JAK3 but also in the upstream signaling components gamma-creceptor chain and IL7 receptor account in aggregate for ˜70% of casesof human severe combined immunodeficiency ('OShea et al., 2004). Notethat JAK1 cooperates with JAK3 in transducing signals from the gamma-creceptor chain. Tyk2 polymorphisms are seen in systemic lupuserythematosus (SLE) (O'Sullivan et al, 2007, Mol. Immunol.44(10):2497-506). Hence, targeting the JAK family may provide atherapeutic opportunity in the immuno-inflammation area.

The current therapies are not satisfactory and therefore there remains aneed to identify further compounds that may be of use in the treatmentof inflammatory conditions, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6. The present invention therefore provides acompound, methods for its manufacture and pharmaceutical compositionscomprising the compound of the invention together with a suitablepharmaceutical carrier. In particular, the compound exhibits highpotency and selectivity for JAK1 over other JAK family members inaddition to 385 kinase and non-kinase targets. Additionally, dataindicates that the compound has a good safety margin. Therefore it isconcluded that the present invention offers a novel opportunity fortreating JAK1 mediated diseases, in particular inflammatory diseasessuch as SLE (systemic lupus erythematosus) and RA.

The present invention also provides for the use of the compound of theinvention in the preparation of a medicament for the treatment of thesediseases and conditions.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that inhibitors of JAK,in particular JAK1 are useful for the treatment of inflammatoryconditions, autoimmune diseases, proliferative diseases, transplantationrejection, diseases involving impairment of cartilage turnover,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6. The present invention also provides methods forthe production of these compounds, pharmaceutical compositionscomprising these compounds and methods for treating inflammatoryconditions, autoimmune diseases, proliferative diseases, transplantationrejection, diseases involving impairment of cartilage turnover,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6 by administering the compound of the invention.

Accordingly, in a first aspect of the invention, a compound of theinvention is disclosed having a Formula I:

The compound of the invention is a novel inhibitor of JAK that exhibitsa high potency and selectivity for JAK1 over other JAK family membersand 385 kinase and non-kinase targets as well as showing a good safetymargin. The use of a compound with this profile may result in advantagesin the treatment of inflammatory diseases, in particular SLE and RA dueto a lower incidence of off-target effects.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising the compound of the invention, and apharmaceutical carrier, excipient or diluent. Moreover, the compound ofthe present invention useful in the pharmaceutical compositions andtreatment methods disclosed herein, is pharmaceutically acceptable asprepared and used. In this aspect of the invention, the pharmaceuticalcomposition may additionally comprise further active ingredientssuitable for use in combination with the compound of the invention.

In a further aspect of the invention, this invention provides a methodof treating a mammal susceptible to or afflicted with a condition fromamong those listed herein, and particularly, such condition as may beassociated with aberrant JAK activity, for example inflammatoryconditions, autoimmune diseases, proliferative diseases, transplantationrejection, diseases involving impairment of cartilage turnover,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6, which method comprises administering atherapeutically effective amount of the compound of the invention, orone or more of the pharmaceutical compositions herein described. In aspecific aspect, this invention provides a method of treating a mammalsusceptible to or afflicted with a condition as may be associated withaberrant JAK1 activity, in particular inflammatory conditions,proliferative diseases and diseases involving impairment of cartilageturnover.

In a further aspect, the present invention provides the compound of theinvention for use in the treatment or prevention of a condition selectedfrom those listed herein, particularly such conditions as may beassociated with aberrant JAK activity such as inflammatory conditions,autoimmune diseases, proliferative diseases, transplantation rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6. In a specific aspect, the present invention provides thecompound of the invention for use in the treatment or prevention ofconditions associated with aberrant JAK1 activity, in particularinflammatory conditions, proliferative diseases and diseases involvingimpairment of cartilage turnover.

In yet another method of treatment aspect, this invention provides amethod for treating a mammal susceptible to or afflicted with acondition that is causally related to abnormal JAK activity as describedherein, which method comprises administering an effectivecondition-treating or condition-preventing amount of one or more of thepharmaceutical compositions or the compound of the invention hereindescribed. In a specific embodiment the abnormal JAK activity isabnormal JAK1 activity.

In a further aspect, the present invention provides the compound of theinvention for use in the treatment or prevention of a condition that iscausally related to abnormal JAK activity. In a specific embodiment theabnormal JAK activity is abnormal JAK1 activity.

In additional aspects, this invention provides methods for synthesizingthe compound of the invention, with representative synthetic protocolsand pathways disclosed later on herein.

Accordingly, it is a principal object of this invention to provide acompound, which can modify the activity of JAK and thus prevent or treatany conditions that may be causally related thereto. In particular anyconditions which may be causally related to the activity of JAK1.

It is further an object of this invention to provide a compound that cantreat or alleviate conditions or diseases or symptoms of same, such asinflammatory conditions, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and diseases associatedwith hypersecretion of IL6, that may be causally related to the activityof JAK. In particular disease or condition which may be causally relatedto the activity of JAK1.

A still further object of this invention is to provide pharmaceuticalcompositions that may be used in the treatment or prevention of avariety of disease states, including the diseases associated with JAKactivity such as inflammatory conditions, autoimmune diseases,proliferative diseases, transplantation rejection, diseases involvingimpairment of cartilage turnover, congenital cartilage malformations,and diseases associated with hypersecretion of IL6. In particularconditions associated with JAK1 activity.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

When describing the invention, which may include compounds,pharmaceutical compositions containing such compounds and methods ofusing such compounds and compositions, the following terms, if present,have the following meanings unless otherwise indicated. It should alsobe understood that when described herein any of the moieties definedforth below may be substituted with a variety of substituents, and thatthe respective definitions are intended to include such substitutedmoieties within their scope as set out below. Unless otherwise stated,the term “substituted” is to be defined as set out below. It should befurther understood that the terms “groups” and “radicals” can beconsidered interchangeable when used herein.

The articles “a” and “an” may be used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example “an analogue” means one analogue or more than oneanalogue.

‘Pharmaceutically acceptable’ means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

‘Pharmaceutically acceptable salt’ refers to a salt of the compound ofthe invention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term“pharmaceutically acceptable cation” refers to an acceptable cationiccounter-ion of an acidic functional group. Such cations are exemplifiedby sodium, potassium, calcium, magnesium, ammonium, tetraalkylammoniumcations, and the like.

‘Pharmaceutically acceptable vehicle’ refers to a diluent, adjuvant,excipient or carrier with which the compound of the invention isadministered.

‘Prodrugs’ refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups and become by solvolysisor under physiological conditions the compounds of the invention whichare pharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like.

‘Solvate’ refers to forms of the compound that are associated with asolvent, usually by a solvolysis reaction. This physical associationincludes hydrogen bonding. Conventional solvents include water, ethanol,acetic acid and the like. The compounds of the invention may be preparede.g. in crystalline form and may be solvated or hydrated. Suitablesolvates include pharmaceutically acceptable solvates, such as hydrates,and further include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. ‘Solvate’ encompasses bothsolution-phase and isolable solvates. Representative solvates includehydrates, ethanolates and methanolates.

‘Subject’ includes humans. The terms ‘human’, ‘patient’ and ‘subject’are used interchangeably herein.

‘Therapeutically effective amount’ means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated.

‘Preventing’ or ‘prevention’ refers to a reduction in risk of acquiringor developing a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a subject that may beexposed to a disease-causing agent, or predisposed to the disease inadvance of disease onset.

The term ‘prophylaxis’ is related to ‘prevention’, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

‘Treating’ or ‘treatment’ of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting thedisease or reducing the manifestation, extent or severity of at leastone of the clinical symptoms thereof). In another embodiment ‘treating’or ‘treatment’ refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,‘treating’ or ‘treatment’ refers to modulating the disease or disorder,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In a further embodiment, “treating” or “treatment” relates to slowingthe progression of the disease.

As used herein the term ‘inflammatory condition(s)’ refers to the groupof conditions including, rheumatoid arthritis, osteoarthritis, juvenileidiopathic arthritis, psoriasis, allergic airway disease (e.g. asthma,rhinitis), inflammatory bowel diseases (e.g. Crohn's disease, colitis),endotoxin-driven disease states (e.g. complications after bypass surgeryor chronic endotoxin states contributing to e.g. chronic cardiacfailure), and related diseases involving cartilage, such as that of thejoints. Partcicularly the term refers to rheumatoid arthritis,osteoarthritis, allergic airway disease (e.g. asthma) and inflammatorybowel diseases.

As used herein the term ‘autoimmune disease(s)’ refers to the group ofdiseases including obstructive airways disease, including conditionssuch as COPD, asthma (e.g intrinsic asthma, extrinsic asthma, dustasthma, infantily asthma) particularly chronic or inveterate asthma (forexample late asthma and airway hyperreponsiveness), bronchitis,including bronchial asthma, systemic lupus erythematosus (SLE), multiplesclerosis, type I diabetes mellitus and complications associatedtherewith, atopic eczema (atopic dermatitis), contact dermatitis andfurther eczematous dermatitis, inflammatory bowel disease (e.g. Crohn'sdisease and ulcerative colitis), atherosclerosis and amyotrophic lateralsclerosis. Particularly the term refers to COPD, asthma, systemic lupuserythematosis, type I diabetes mellitus and inflammatory bowel disease.

As used herein the term ‘proliferative disease(s)’ refers to conditionssuch as cancer (e.g. uterine leiomyosarcoma or prostate cancer),myeloproliferative disorders (e.g. polycythemia vera, essentialthrombocytosis and myelofibrosis), leukemia (e.g. acute myeloidleukaemia and acute lymphoblastic leukemia), multiple myeloma,psoriasis, restenosis, sclerodermitis or fibrosis. In particular theterm refers to cancer, leukemia, multiple myeloma and psoriasis.

As used herein, the term ‘cancer’ refers to a malignant or benign growthof cells in skin or in body organs, for example but without limitation,breast, prostate, lung, kidney, pancreas, stomach or bowel. A cancertends to infiltrate into adjacent tissue and spread (metastasise) todistant organs, for example to bone, liver, lung or the brain. As usedherein the term cancer includes both metastatic tumour cell types, suchas but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma,rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such asbut not limited to, colorectal cancer, prostate cancer, small cell lungcancer and non-small cell lung cancer, breast cancer, pancreatic cancer,bladder cancer, renal cancer, gastric cancer, glioblastoma, primaryliver cancer, ovarian cancer, prostate cancer and uterineleiomyosarcoma.

As used herein the term ‘leukemia’ refers to neoplastic diseases of theblood and blood forming organs. Such diseases can cause bone marrow andimmune system dysfunction, which renders the host highly susceptible toinfection and bleeding. In particular the term leukemia refers to acutemyeloid leukaemia (AML) and acute lymphoblastic leukemia (ALL).

As used herein the term ‘transplantation rejection’ refers to the acuteor chronic rejection of cells, tissue or solid organ allo- or xenograftsof e.g. pancreatic islets, stem cells, bone marrow, skin, muscle,corneal tissue, neuronal tissue, heart, lung, combined heart-lung,kidney, liver, bowel, pancreas, trachea or oesophagus, orgraft-versus-host diseases.

As used herein the term ‘diseases involving impairment of cartilageturnover’ includes conditions such as osteoarthritis, psoriaticarthritis, juvenile rheumatoid arthritis, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia,osteochondritis, neurogenic or neuropathic arthritis, arthropathy,endemic forms of arthritis like osteoarthritis deformans endemica,Mseleni disease and Handigodu disease; degeneration resulting fromfibromyalgia, systemic lupus erythematosus, scleroderma and ankylosingspondylitis.

As used herein the term ‘congenital cartilage malformation(s)’ includesconditions such as hereditary chondrolysis, chondrodysplasias andpseudochondrodysplasias, in particular, but without limitation,microtia, anotia, metaphyseal chondrodysplasia, and related disorders.

As used herein the term ‘disease(s) associated with hypersecretion ofIL6’ includes conditions such as Castleman's disease, multiple myeloma,psoriasis, Kaposi's sarcoma and/or mesangial proliferativeglomerulonephritis.

As used herein the term “JAK” relates to the family of Janus kinases(JAKs) which are cytoplasmic tyrosine kinases that transduce cytokinesignaling from membrane receptors to STAT transcription factors. FourJAK family members are described, JAK1, JAK2, JAK3 and TYK2 and the termJAK may refer to all the JAK family members collectively or one or moreof the JAK family members as the context indicates.

‘Compound(s) of the invention’, and equivalent expressions, are meant toembrace the compound of the Formula as herein described, whichexpression includes the pharmaceutically acceptable salts, and thesolvates, e.g., hydrates, and the solvates of the pharmaceuticallyacceptable salts where the context so permits. Similarly, reference tointermediates, whether or not they themselves are claimed, is meant toembrace their salts, and solvates, where the context so permits.

When ranges are referred to herein, for example but without limitation,C₁-C₈ alkyl, the citation of a range should be considered arepresentation of each member of said range.

Other derivatives of the compounds of this invention have activity inboth their acid and acid derivative forms, but in the acid sensitiveform often offers advantages of solubility, tissue compatibility, ordelayed release in the mammalian organism (see, Bundgard, H., Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs includeacid derivatives well know to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acid with a suitablealcohol, or amides prepared by reaction of the parent acid compound witha substituted or unsubstituted amine, or acid anhydrides, or mixedanhydrides. Simple aliphatic or aromatic esters, amides and anhydridesderived from acidic groups pendant on the compounds of this inventionare particularly useful prodrugs. In some cases it is desirable toprepare double ester type prodrugs such as (acyloxy)alkyl esters or((alkoxycarbonyl)oxy)alkylesters. Particular such prodrugs are the C₁ toC_(s) alkyl, C₂-C₈ alkenyl, aryl, C₇-C₁₂ substituted aryl, and C₇-C₁₂arylalkyl esters of the compounds of the invention.

As used herein, the term ‘isotopic variant’ refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an ‘isotopic variant’ of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e. and carbon-14, i.e. ¹⁴Care particularly useful for this purpose in view of their ease ofincorporation and ready means of detection. Further, compounds may beprepared that are substituted with positron emitting isotopes, such as¹¹C, ¹⁸F, ¹⁵O and a ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy.

All isotopic variants of the compound provided herein, radioactive ornot, are intended to be encompassed within the scope of the invention.

Stereoisomers that are not mirror images of one another are termed‘diastereomers’ and those that are non-superimposable mirror images ofeach other are termed ‘enantiomers’. When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a ‘racemic mixture’.

‘Tautomers’ refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of π electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro- forms of phenylnitromethane, that arelikewise formed by treatment with acid or base.

Tautomeric forms may be relevant to the attainment of the optimalchemical reactivity and biological activity of a compound of interest.

The compound of the invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)— or(S)— stereoisomers or as mixtures thereof.

Unless indicated otherwise, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art.

The Compound

The present invention is based on the discovery that inhibitors of JAKare useful for the treatment of inflammatory conditions, autoimmunediseases, proliferative diseases, transplantation rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and diseases associated with hypersecretion of IL6. Thepresent invention also provides methods for the production of thecompound, pharmaceutical compositions comprising the compound andmethods for treating diseases involving cartilage degradation, boneand/or joint degradation and/or inflammation by administering thecompound of the invention. The present compound is an inhibitor ofmembers of the JAK family; specifically it inhibits the activity ofJAK1, JAK2, JAK3 and TYK2. In particular it inhibits the activity ofJAK1.

Accordingly, in a first aspect of the invention, a compound of theinvention is disclosed having a Formula I:

The compound of the invention is cyclopropanecarboxylic acid{5-[4-(3,3-dimethyl-azetidine-1-carbonyl)-phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl}-amide.

In one embodiment the compound of the invention is not an isotopicvariant.

In one aspect the compound of the invention is present as the free base.

In one aspect the compound of the invention is a pharmaceuticallyacceptable salt.

In one aspect the compound of the invention is a solvate of thecompound.

In one aspect the compound of the invention is a solvate of apharmaceutically acceptable salt of the compound.

In certain aspects, the present invention provides prodrugs andderivatives of the compound according to the formula above. Prodrugs arederivatives of the compounds of the invention, which have metabolicallycleavable groups and become by solvolysis or under physiologicalconditions the compounds of the invention, which are pharmaceuticallyactive, in vivo. Such examples include, but are not limited to, cholineester derivatives and the like, N-alkylmorpholine esters and the like.

Other derivatives of the compound of the invention have activity in boththeir acid and acid derivative forms, but the acid sensitive form oftenoffers advantages of solubility, tissue compatibility, or delayedrelease in the mammalian organism (see, Bundgard, H., Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs includeacid derivatives well know to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acid with a suitablealcohol, or amides prepared by reaction of the parent acid compound witha substituted or unsubstituted amine, or acid anhydrides, or mixedanhydrides. Simple aliphatic or aromatic esters, amides and anhydridesderived from acidic groups pendant on the compounds of this inventionare preferred prodrugs. In some cases it is desirable to prepare doubleester type prodrugs such as (acyloxy)alkyl esters or((alkoxycarbonyl)oxy)alkylesters. Particularly useful are the C₁ to C₈alkyl, C₂-C₈ alkenyl, aryl, C₇-C₁₂ substituted aryl, and C₇-C₁₂arylalkyl esters of the compounds of the invention.

Pharmaceutical Compositions

When employed as a pharmaceutical, the compound of the invention istypically administered in the form of a pharmaceutical composition. Suchcompositions can be prepared in a mariner well known in thepharmaceutical art and comprise at least one active compound. Generally,the compound of the invention is administered in a pharmaceuticallyeffective amount. The amount of the compound actually administered willtypically be determined by a physician, in the light of the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the actual compound-administered, the age, weight,and response of the individual patient, the severity of the patient'ssymptoms, and the like.

The pharmaceutical compositions of the invention can be administered bya variety of routes including oral, rectal, transdermal, subcutaneous,intra-articular, intravenous, intramuscular, and intranasal. Dependingon the intended route of delivery, the compound of this invention ispreferably formulated as either injectable or oral compositions or assalves, as lotions or as patches all for transdermal administration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient, vehicle orcarrier. Typical unit dosage forms include prefilled, premeasuredampules or syringes of the liquid compositions or pills, tablets,capsules or the like in the case of solid compositions. In suchcompositions, the compound of the invention is usually a minor component(from about 0.1 to about 50% by weight or preferably from about 1 toabout 40% by weight) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

Liquid forms suitable for oral administration may include a suitableaqueous or nonaqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active compound in such compositions istypically a minor component, often being from about 0.05 to 10% byweight with the remainder being the injectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as a ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope of this invention.

The compound of the invention can also be administered by a transdermaldevice. Accordingly, transdermal administration can be accomplishedusing a patch either of the reservoir or porous membrane type, or of asolid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, MackPublishing Company, Easton, Pa., which is incorporated herein byreference.

The compound of the invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can be foundin Remington's Pharmaceutical Sciences.

The following formulation examples illustrate representativepharmaceutical compositions that may be prepared in accordance with thisinvention. The present invention, however, is not limited to thefollowing pharmaceutical compositions.

Formulation 1—Tablets

The compound of the invention may be admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate may be added as a lubricant. The mixture may beformed into 240-270 mg tablets (80-90 mg of active amide compound pertablet) in a tablet press.

Formulation 2—Capsules

The compound of the invention may be admixed as a dry powder with astarch diluent in an approximate 1:1 weight ratio. The mixture may befilled into 250 mg capsules (125 mg of active amide compound percapsule).

Formulation 3—Liquid

The compound of the invention (125 mg), may be admixed with sucrose(1.75 g) and xanthan gum (4 mg) and the resultant mixture may beblended, passed through a No. 10 mesh U.S. sieve, and then mixed with apreviously made solution of microcrystalline cellulose and sodiumcarboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10mg), flavor, and color may be diluted with water and added withstirring. Sufficient water may then be added with stirring. Sufficientwater may be then added to produce a total volume of 5 mL.

Formulation 4—Tablets

The compound of the invention may be admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnesium stearate may be added as a lubricant. The mixture is formedinto 450-900 mg tablets (150-300 mg of active amide compound) in atablet press.

Formulation 5—Injection

The compound of the invention may be dissolved or suspended in abuffered sterile saline injectable aqueous medium to a concentration ofapproximately 5 mg/mL.

Formulation 6—Topical

Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted atabout 75° C. and then a mixture of the compound of the invention (50 g)methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate(10 g), and propylene glycol (120 g) dissolved in water (about 370 g)may be added and the resulting mixture may be stirred until it congeals.

Methods of Treatment

The compound of the invention may be used as a therapeutic agent for thetreatment of conditions in mammals that are causally related orattributable to aberrant activity of JAK. In particular, conditionsrelated to aberrant activity of JAK1. Accordingly, the compound andpharmaceutical compositions of the invention find use as therapeuticsfor preventing and/or treating inflammatory conditions, autoimmunediseases, proliferative diseases, transplantation rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and diseases associated with hypersecretion of IL6 inmammals including humans.

In additional method of treatment aspects, this invention providesmethods of treating a mammal susceptible to or afflicted with aninflammatory condition. The methods comprise administering an effectivecondition-treating or condition-preventing amount of one or more of thepharmaceutical compositions or the compound of the invention hereindescribed. In a specific embodiment, the inflammatory condition isselected from rheumatoid arthritis, osteoarthritis, allergic airwaydisease (e.g. asthma) and inflammatory bowel diseases.

In another aspect the present invention provides the compound of theinvention for use in the treatment, prevention or prophylaxis of aninflammatory condition. In a specific embodiment, the inflammatorycondition is selected from rheumatoid arthritis, osteoarthritis,allergic airway disease (e.g. asthma) and inflammatory bowel diseases.

In additional method of treatment aspects, this invention providesmethods of treating a mammal susceptible to or afflicted with anautoimmune disease. The methods comprise administering an effectivecondition-treating or condition-preventing amount of one or more of thepharmaceutical compositions or the compound of the invention hereindescribed. In a specific embodiment, the autoimmune disease is selectedfrom COPD, asthma, systemic lupus erythematosis, type I diabetesmellitus and inflammatory bowel disease.

In another aspect the present invention provides the compound of theinvention for use in the treatment, prevention or prophylaxis of anautoimmune disease. In a specific embodiment, the autoimmune disease isselected from COPD, asthma, systemic lupus erythematosis, type Idiabetes mellitus and inflammatory bowel disease.

In further method of treatment aspects, this invention provides methodsof treating a mammal susceptible to or afflicted with a proliferativedisease, in particular cancer (e.g. solid tumors such as uterineleiomyosarcoma or prostate cancer), leukemia (e.g. AML or ALL), multiplemyeloma and/or psoriasis. The methods comprise administering aneffective condition-treating or condition-preventing amount of one ormore of the pharmaceutical compositions or the compound of the inventionherein described.

In another aspect the present invention provides the compound of theinvention for use in the treatment, prevention or prophylaxis of aproliferative disease, in particular cancer (e.g. solid tumors such asuterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML or ALL),multiple myeloma and/or psoriasis.

In further method of treatment aspects, this invention provides methodsof treating a mammal susceptible to or afflicted with transplantationrejection. The methods comprise administering an effectivecondition-treating or condition-preventing amount of one or more of thepharmaceutical compositions or the compound of the invention hereindescribed. In a specific embodiment, the invention provides methods oftreating organ transplant rejection.

In another aspect the present invention provides the compound of theinvention for use in the treatment, prevention or prophylaxis oftransplantation rejection. In a specific embodiment, the inventionprovides methods of treating organ transplant rejection.

In a method of treatment aspect, this invention provides a method oftreatment, prevention or prophylaxis in a mammal susceptible to orafflicted with diseases involving impairment of cartilage turnover. Themethods comprise administering an effective condition-treating orcondition-preventing amount of one or more of the pharmaceuticalcompositions or the compound of the invention herein described.

In another aspect the present invention provides the compound of theinvention for use in the treatment, prevention or prophylaxis ofdiseases involving impairment of cartilage turnover.

The present invention also provides a method of treatment of congenitalcartilage malformations. The methods comprise administering an effectivecondition-treating or condition-preventing amount of one or more of thepharmaceutical compositions or the compound of the invention hereindescribed.

In another aspect the present invention provides the compound of theinvention for use in the treatment, prevention or prophylaxis ofcongenital cartilage malformations.

In further method of treatment aspects, this invention provides methodsof treating a mammal susceptible to or afflicted with diseasesassociated with hypersecretion of IL6, in particular Castleman's diseaseor mesangial proliferative glomerulonephritis. The methods compriseadministering an effective condition-treating or condition-preventingamount of one or more of the pharmaceutical compositions or the compoundof the invention herein described.

In another aspect the present invention provides the compound of theinvention for use in the treatment, prevention or prophylaxis ofdiseases associated with hypersecretion of IL6, in particularCastleman's disease or mesangial proliferative glomerulonephritis.

As a further aspect of the invention there is provided the compound ofthe invention for use as a pharmaceutical especially in the treatment orprevention of the aforementioned conditions and diseases. Also providedherein is the use of the compound in the manufacture of a medicament forthe treatment or prevention of one of the aforementioned conditions anddiseases.

A particular regimen of the present method comprises the administrationto a subject in suffering from an inflammatory condition, of aneffective amount of the compound of the invention for a period of timesufficient to reduce the level of inflammation in the subject, andpreferably terminate, the processes responsible for said inflammation. Aspecial embodiment of the method comprises administering of an effectiveamount of the compound of the invention to a subject patient sufferingfrom or susceptible to the development of rheumatoid arthritis, for aperiod of time sufficient to reduce or prevent, respectively,inflammation in the joints of said patient, and preferably terminate,the processes responsible for said inflammation.

A further particular regimen of the present method comprises theadministration to a subject suffering from diseases involving impairmentof cartilage turnover (e.g. osteoarthritis) of an effective amount ofthe compound of the invention for a period of time sufficient to reduceand preferably terminate, the self-perpetuating processes responsiblefor said degradation. A particular embodiment of the method comprisesadministering of an effective amount of the compound of the invention toa subject patient suffering from or susceptible to the development ofosteoarthritis, for a period of time sufficient to reduce or prevent,respectively, cartilage degradation in the joints of said patient, andpreferably terminate, the self-perpetuating processes responsible forsaid degradation. In a particular embodiment said compound may exhibitcartilage anabolic and/or anti-catabolic properties.

Injection dose levels range from about 0.1 mg/kg/hour to at least 10mg/kg/hour, all for from about 1 to about 120 hours and especially 24 to96 hours. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kgor more may also be administered to achieve adequate steady statelevels. The maximum total dose is not expected to exceed about 2 g/dayfor a 40 to 80 kg human patient.

For the prevention and/or treatment of long-term conditions, such asdegenerative conditions, the regimen for treatment usually stretchesover many months or years so oral dosing is preferred for patientconvenience and tolerance. With oral dosing, one to five and especiallytwo to four and typically three oral doses per day are representativeregimens. Using these dosing patterns, each dose provides from about0.01 to about 20 mg/kg of the compound of the invention, with particulardoses each providing from about 0.1 to about 10 mg/kg and especiallyabout 1 to about 5 mg/kg.

Transdermal doses are generally selected to provide similar or lowerblood levels than are achieved using injection doses.

When used to prevent the onset of an inflammatory condition, thecompound of the invention will be administered to a patient at risk fordeveloping the condition, typically on the advice and under thesupervision of a physician, at the dosage levels described above.Patients at risk for developing a particular condition generally includethose that have a family history of the condition, or those who havebeen identified by genetic testing or screening to be particularlysusceptible to developing the condition.

The compound of the invention can be administered as the sole activeagent or it can be administered in combination with other therapeuticagents, including other compounds that demonstrate the same or a similartherapeutic activity, and that are determined to be safe and efficaciousfor such combined administration. In a specific embodiment,co-administration of two (or more) agents allows for significantly lowerdoses of each to be used, thereby reducing the side effects seen.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention of aninflammatory condition; particular agents include, but are not limitedto, immunoregulatory agents e.g. azathioprine, corticosteroids (e.g.prednisolone or dexamethasone), cyclophosphamide, cyclosporin A,tacrolimus, Mycophenolate Mofetil, muromonab-CD3 (OKT3, e.g.Orthocolone®), ATG, aspirin, acetaminophen, ibuprofen, naproxen, andpiroxicam.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention ofarthritis (e.g. rheumatoid arthritis); particular agents include but arenot limited to analgesics, non-steroidal anti-inflammatory drugs(NSAIDS), steroids, synthetic DMARDS (for example but without limitationmethotrexate, leflunomide, sulfasalazine, auranofin, sodiumaurothiomalate, penicillamine, chloroquine, hydroxychloroquine,azathioprine, and cyclosporin), and biological DMARDS (for example butwithout limitation Infliximab, Etanercept, Adalimumab, Rituximab, andAbatacept).

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention ofproliferative disorders; particular agents include but are not limitedto: methotrexate, leukovorin, adriamycin, prenisone, bleomycin,cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine,vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrolacetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g.Herceptin™), capecitabine, raloxifene hydrochloride, EGFR inhibitors(e.g. Iressa®, Tareeva™, Erbitux™), VEGF inhibitors (e.g. Avastin™),proteasome inhibitors (e.g. Velcade™), Glivec® and hsp90 inhibitors(e.g. 17-AAG). Additionally, a compound of the invention may beadministered in combination with other therapies including, but notlimited to, radiotherapy or surgery. In a specific embodiment theproliferative disorder is selected from cancer, myeloproliferativedisease or leukaemia.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention ofautoimmune diseases; particular agents include but are not limited to:glucocorticoids, cytostatic agents (e.g. purine analogs), alkylatingagents, (e.g nitrogen mustards (cyclophosphamide), nitrosoureas,platinum compounds, and others), antimetabolites (e.g. methotrexate,azathioprine and mercaptopurine), cytotoxic antibiotics (e.g.dactinomycin anthracyclines, mitomycin C, bleomycin, and mithramycin),antibodies (e.g., anti-CD20, anti-CD25 or anti-CD3 (OTK3) monoclonalantibodies, Atgam® and Thymoglobuline®), cyclosporin, tacrolimus,rapamycin (sirolimus), interferons (e.g. IFN-β), TNF binding proteins(e.g. infliximab (Remicade™), etanercept (Enbrel™), or adalimumab(Humira™)), mycophenolate, Fingolimod, and Myriocin.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention oftransplantation rejection; particular agents include but are not limitedto: calcineurin inhibitors (e.g. cyclosporin or tacrolimus (FK506)),mTOR inhibitors (e.g. sirolimus, everolimus), anti-proliferatives (e.g.azathioprine, mycophenolic acid), corticosteroids (e.g. prednisolone,hydrocortisone), Antibodies (e.g. monoclonal anti-IL-2Rα receptorantibodies, basiliximab, daclizumab), polyclonal anti-T-cell antibodies(e.g. anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG)).

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention of asthmaand/or rhinitis and/or COPD; particular agents include but are notlimited to: beta₂-adrenoceptor agonists (e.g. salbutamol, levalbuterol,terbutaline and bitolterol), epinephrine (inhaled or tablets),anticholinergics (e.g. ipratropium bromide), glucocorticoids (oral orinhaled) Long-acting β₂-agonists (e.g. salmeterol, formoterol,bambuterol, and sustained-release oral albuterol), combinations ofinhaled steroids and long-acting bronchodilators (e.g.fluticasone/salmeterol, budesonide/formoterol), leukotriene antagonistsand synthesis inhibitors (e.g. montelukast, zafirlukast and zileuton),inhibitors of mediator release (e.g. cromoglycate and ketotifen),biological regulators of IgE response (e.g. omalizumab), antihistamines(e.g. ceterizine, cinnarizine, fexofenadine), and vasoconstrictors (e.g.oxymethazoline, xylomethazoline, nafazoline and tramazoline).

Additionally, the compound of the invention may be administered incombination with emergency therapies for asthma and/or COPD, suchtherapies include oxygen or heliox administration, nebulized salbutamolor terbutaline (optionally combined with an anticholinergic (e.g.ipratropium), systemic steroids (oral or intravenous, e.g. prednisone,prednisolone, methylprednisolone, dexamethasone, or hydrocortisone),intravenous salbutamol, non-specific beta-agonists, injected or inhaled(e.g. epinephrine, isoetharine, isoproterenol, metaproterenol),anticholinergics (IV or nebulized, e.g. glycopyrrolate, atropine,ipratropium), methylxanthines (theophylline, aminophylline,bamiphylline), inhalation anesthetics that have a bronchodilatory effect(e.g. isoflurane, halothane, enflurane), ketamine, and intravenousmagnesium sulfate.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention ofirritable bowel disease (IBD); particular agents include but are notlimited to: glucocorticoids (e.g. prednisone, budesonide) syntheticdisease modifying, immunomodulatory agents (e.g. methotrexate,leflunomide, sulfasalazine, mesalazine, azathioprine, 6-mercaptopurineand ciclosporin) and biological disease modifying, immunomodulatoryagents (infliximab, adalimumab, rituximab, and abatacept).

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention ofsystemic lupus erythematosus; particular agents include but are notlimited to: disease-modifying antirheumatic drugs (DMARDs) such asantimalarials (e.g. plaquenil, hydroxychloroquine), immunosuppressants(e.g. methotrexate and azathioprine), cyclophosphamide and mycophenolicacid; immunosuppressive drugs and analgesics, such as nonsteroidalanti-inflammatory drugs, opiates (e.g. dextropropoxyphene andco-codamol), opioids (e.g. hydrocodone, oxycodone, MS Contin, ormethadone) and the fentanyl duragesic transdermal patch.

In one embodiment, the compound of the invention is co-administered withanother therapeutic agent for the treatment and/or prevention ofpsoriasis; particular agents include but are not limited to: topicaltreatments such as bath solutions, moisturizers, medicated creams andointments containing coal tar, dithranol (anthralin), corticosteroidslike desoximetasone (Topicort™), fluocinonide, vitamin D₃ analogues (forexample, calcipotriol), Argan oil and retinoids (etretinate, acitretin,tazarotene), systemic treatments such as methotrexate, cyclosporine,retinoids, tioguanine, hydroxyurea, sulfasalazine, mycophenolatemofetil, azathioprine, tacrolimus, fumaric acid esters or biologics suchas Amevive™, Enbrel™, Humira™, Remicade™, Raptiva™ and ustckinumab (aIL-12 and IL-23 blocker). Additionally, a compound of the invention maybe administered in combination with other therapies including, but notlimited to phototherapy, or photochemotherapy (e.g. psoralen andultraviolet A phototherapy (PUVA)).

By co-administration is included any means of delivering two or moretherapeutic-agents to the patient as part of the same treatment regime,as will be apparent to the skilled person. Whilst the two or more agentsmay be administered simultaneously in a single formulation this is notessential. The agents may be administered in different formulations andat different times.

General Synthetic Procedures General

The compound of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and P. G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, New York, 1991, andreferences cited therein.

The following methods are presented with details as to the preparationof representative bicycloheteroaryls that have been listed hereinabove.The compound of the invention may be prepared from known or commerciallyavailable starting materials and reagents by one skilled in the art oforganic synthesis.

All reagents were of commercial grade and were used as received withoutfurther purification, unless otherwise stated. Commercially availableanhydrous solvents were used for reactions conducted under inertatmosphere. Reagent grade solvents were used in all other cases, unlessotherwise specified. Column chromatography was performed on silica gel60 (35-70 μm). Thin layer chromatography was carried out usingpre-coated silica gel F-254 plates (thickness 0.25 mm). ¹H NMR spectrawere recorded on a Bruker DPX 400 NMR spectrometer (400 MHz). Chemicalshifts (6) for ¹H NMR spectra are reported in parts per million (ppm)relative to tetramethylsilane (δ 0.00) or the appropriate residualsolvent peak, i.e. CHCl₃ (δ 7.27), as internal reference. Multiplicitiesare given as singlet (s), doublet (d), triplet (t), quartet (q),multiplet (m) and broad (br). Coupling constants (J) are given in Hz.Electrospray MS spectra were obtained on a Micromass platform LC/MSspectrometer. Column Used for all LCMS analysis: Waters Acquity UPLC BEHC18 1.7 μm, 2.1 mm TD×50 mm L (Part No. 186002350)). Preparative HPLC:Waters XBridge Prep C18 5 μm ODB 19 mm ID×100 mm L (Part No. 186002978).All the methods are using MeCN/H₂O gradients. H₂O contains either 0.1%TFA or 0.1% NH₃.

The following is a list of abbreviations used in the experimentalsection:

DCM Dichloromethane DiPEA N,N-diisopropylethylamine MeCN AcetonitrileBOC tert-Butyloxy-carbonyl MF N,N-dimethylformamide Cat. Catalyticamount TFA Trifluoroacetic acid THF Tetrahydrofuran NMR Nuclear MagneticResonnance DMSO Dimethylsulfoxide LC-MS Liquid Chromatography-MassSpectrometry Ppm part-per-million Pd/C Palladium on Charcoal 10% PMBPara-methoxy-benzyl PyBOP benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluoroborate EtOAc ethyl acetate APCIatmospheric pressure chemical ionization Rt retention time s singlet brs broad singlet m multiplet min minute mL milliliter μL microliter ggram mg milligram PdCl₂dppf [1,1′-Bis(diphenylphosphino)ferro- cene]dichloropalladium(II) TEA Triethylamine MMP Matrix Metallo ProteinaseNHAC Normal Human Articular Chondrocytes shRNA short hairpin RNA RNARibonucleic acid Ad-Si RNA Adenoviral encoded siRNA PBST Phosphatebuffered saline with Tween 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl,135 mM NaCl, 0.05% Tween 20, pH 7.4 APMA 4-aminophenylmercuric acetateDMEM Dulbecco's Modified Eagle Medium FBS Fetal bovine serum hCAR humancellular adenovirus receptor 3- MOI multiplicity of infection of 3 dNTPdeoxyribonucleoside triphosphate QPCR quantitative polymerase chainreaction cDNA copy deoxyribonucleic acid GAPDH Glyceraldehyde phosphatedehydrogenase

Synthetic Preparation of the Compound of the Invention

The compound according to the invention can be produced according to thefollowing scheme.

General Synthetic Method

wherein Ar represents phenyl-L1-heterocycloalkyl, where L1 is —CO— andthe heterocycloalkyl group is optionally substituted.

General 1.1.1 1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea (2)

To a solution of 2-amino-6-bromopyridine (1) (253.8 g, 1.467 mol) in DCM(2.5 L) cooled to 5° C. is added ethoxycarbonyl isothiocyanate (173.0mL, 1.467 mol) dropwise over 15 min. The reaction mixture is thenallowed to warm to room temp. (20° C.) and stirred for 16 h. Evaporationin vacuo gives a solid which may be collected by filtration, thoroughlywashed with petrol (3×600 mL) and air-dried to afford (2). The thioureamay be used as such for the next step without any purification. ¹H (400MHz, CDCl₃) δ 12.03 (1H, br s, NH), 8.81 (1H, d, J=7.8 Hz, H-3), 8.15(1H, br s, NH), 7.60 (1H, t, J=8.0 Hz, H-4), 7.32 (1H, dd, J 7.7 and 0.6Hz, H-5), 4.31 (2H, q, J=7.1 Hz, CH₂), 1.35 (3H, t, J=7.1 Hz, CH₃).

1.1.2 5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (3)

To a suspension of hydroxylamine hydrochloride (101.8 g, 1.465 mol) inEtOH/MeOH (1:1, 900 mL) is added N,N-diisopropylethylamine (145.3 mL,0.879 mol) and the mixture is stirred at room temp. (20° C.) for 1 h.1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea (2) (89.0 g, 0.293 mol)is then added and the mixture slowly heated to reflux (Note: bleachscrubber is required to quench H₂S evolved). After 3 h at reflux, themixture is allowed to cool and filtered to collect the precipitatedsolid. Further product is collected by evaporation in vacuo of thefiltrate, addition of H₂O (250 mL) and filtration. The combined solidsare washed successively with H₂O (250 mL), EtOH/MeOH (1:1, 250 mL) andEt₂O (250 mL) then dried in vacuo to afford the triazolopyridinederivative (3) as a solid. The compound may be used as such for the nextstep without any purification. ¹H (400 MHz, DMSO-d₆) δ 7.43-7.34 (2H, m,2× aromatic-H), 7.24 (1H, dd, J 6.8 and 1.8 Hz, aromatic-H), 6.30 (2H,br, NH₂); m/z 213/215 (1:1, M+H⁺, 100%).

1.1.3 Cyclopropanecarboxylic acid(5-bromo-[1,2,4]-triazolo[1,5-a]pyridin-2-yl)-amide (4)

To a solution of the 2-amino-triazolopyridine (3) (7.10 g, 33.3 mmol) indry CH₃CN (150 mL) at 5° C. is added Et₃N (11.6 mL, 83.3 mmol) followedby cyclopropanecarbonyl chloride (83.3 mmol). The reaction mixture isthen allowed to warm to ambient temperature and stirred until allstarting material (3) is consumed. If required, further Et₃N (4.64 mL,33.3 mmol) and cyclopropanecarbonyl chloride (33.3 mmol) is added toensure complete reaction. Following solvent evaporation in vacuo theresultant residue is treated with 7 N methanolic ammonia solution (50mL) and stirred at ambient temp. (for 1-16 h) to hydrolyse anybis-acylated product. Product isolation is made by removal of volatilesin vacuo followed by trituration with Et₂O (50 mL). The solids arecollected by filtration, washed with H₂O (2×50 mL), acetone (50 mL) andEt₂O (50 mL), then dried in vacuo to give the required bromointermediate (4).

Synthetic Procedure for the Preparation of the Compound of the InventionCompound 1 Step 1: Suzuki Coupling

4-Carboxyphenylboronic acid (3.5 g, 0.021 mol.) was added to a solutionof cyclopropanecarboxylic acid(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide (Intermediate 4, 5 g,0.018 mol) in 1,4-dioxane/water (5:1). K₂CO₃ (5.0 g, 0.036 mol) andPdCl₂dppf (5%) were added to the solution. The resulting mixture wasthen heated by traditional heating in an oil bath at 90° C. for 16 h. 1M HCl solution was added and a precipitate was formed in the acidicsolution. The precipitate was filtered, dried under vacuum to give thetitle compound used in the next step without further purification.

Step 2: Cyclopropanecarboxylic acid{5-[4-(3,3-dimethyl-azetidine-1-carbonyl)-phenyl-1,2,4]-triazolo[1,5-a]pyridin-2-yl}-amide(Compound 1)

EDCI (3.59 g, 0.019 mol), HOBt (2.53 g, 0.019 mol) and DIPEA (4.48 mL)were added to a solution of4-[2-(Cyclopropanecarbonyl-amino)-[1,2,4]triazolo[1,5-a]pyridin-5-yl]-benzoicacid (4 g, 0.012 mol) in DCM (150 mL) at room temperature. The resultingmixture was stirred for 10 min at room temperature. Dimethylazetidinehydrochloride salt (1.64 g, 0.013 mol) was added to the solution and thereaction is stirred for 16 hrs. Water was added to the reaction mixture.The organic layer was separated and washed with 2 N NaOH solution, 2NHCl solution and water. The organic phase was dried over MgSO₄, filteredand evaporated under vacuum. Purification by flash chromatography(eluant: 1:1 petrol/EtOAc to neat EtOAc) afforded cyclopropanecarboxylicacid{5-[4-(3,3-dimethyl-azetidine-1-carbonyl)-phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl}-amide.

The compound of the invention that has been or can be prepared accordingto the synthetic method described herein is listed in Table I below. TheNMR spectral data of the compound of the invention is given in Table II.

TABLE I Mass spectral data of the Compound of the Invention Cpd MS MS #Structures Name MW Mes'd 1

Cyclopropanecarboxylic acid {5-[4-(3,3-dimethyl-azetidine-1-carbonyl)-phenyl]- [1,2,4]triazolo[1,5-a]pyridin-2- yl}-amide 389.46390.0

TABLE II NMR Data of the Compound of the Invention Cpd # NMR data (δ) 1(¹H, DMSO-d6) 11.01 (1H, b, NH), 8.08 (2H, d, ArH), 7.79 (2H, d, ArH),7.73 (1H, d, ArH), 7.72 (1H, s, ArH), 7.35 (1H, dd, ArH), 4.04 (2H, b,CH₂), 3.77 (2H, b, CH₂), 2.02 (1H, b, CH), 1.27 (6H, s, 2xCH₃), 0.81(4H, m, 2xCH₂).

Biological Examples Example 1 In Vitro Assays Example 1.1 JAK1Inhibition Assay

Recombinant human JAK1 (catalytic domain, amino acids 866-1154; catalognumber PV4774) was purchased from Invitrogen. 1 ng of JAK1 was incubatedwith 20 nM Ulight-JAK1(tyr1023) peptide (Perkin Elmer catalog numberTRF0121) in kinase reaction buffer (25 mM MOPS pH6.8, 0.016% Brij-35,8.33 mM MgCl2, 3.33 mM DTT, 7 μM ATP) with or without 4 μL containingtest compound or vehicle (DMSO, 1% final concentration), in a totalvolume of 20 μL, in a white 384 Luminotrac 200 plate (Greiner, catalognumber 781075). After 60 min at room temperature, reactions were stoppedby adding 20 μL/well of detection mixture (1× detection buffer (PerkinElmer, catalog number CR97-100C), 0.5 nM Europium-anti-phosphotyrosine(PT66) (Perkin Elmer, catalog number AD0068), 10 mM EDTA). Readout isperformed using the Envision with excitation at 320 nm and measuringemission at 615 nm (Perkin Elmer). Kinase activity was calculated bysubtracting relative fluorescence units (RFU) obtained in the presenceof a positive control inhibitor (10 μM staurosporine) from RFU obtainedin the presence of vehicle. The ability of a test compound to inhibitthis activity was determined as:

Percentage inhibition=((RFU determined for sample with test compoundpresent−RFU determined for sample with positive control inhibitor)divided by (RFU determined in the presence of vehicle−RFU determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the JAK1 assay and the calculationof the IC₅₀ for the compound. Each compound is routinely tested atconcentration of 20 μM followed by a 1/5 serial dilution, 8 points (20μM-4 μM-800 nM-160 nM-32 nM-6.4 nM-1.28 nM-0.26 nM) in a finalconcentration of 1% DMSO. When potency of compound series increases,more dilutions are prepared and/or the top concentration are lowered(e.g. 5 μM, 1 μM). The data are expressed as the average IC₅₀ from theassays±standard error of the mean.

TABLE III JAK1 IC₅₀ Values of Compound Cpd # JAK1 (nM) 1 6.6 ± 0.7

Example 1.2 JAK1 Ki Determination Assay

For the determination of Ki, different amounts of inhibitor were mixedwith the enzyme and the enzymatic reaction was followed as a function ofATP concentration. The Ki was determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). JAK1(Invitrogen, PV4774) was used at a final concentration of 500 ng/ml. Thesubstrate was Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50 000 (Sigma,P0275) The reaction was performed in 25 mM Hepes pH 7.5; 0.01% Tween20,mM MgCl₂ with varying concentrations of ATP and compound and stopped byaddition of 150 mM phosphoric acid. Measurement of incorporatedphosphate into the substrate polyGT was done by loading the samples on afilter plate (using a harvester, Perkin Elmer) and subsequent washing.Incorporated ³³P in polyGT is measured in a Topcount scintillationcounter after addition of scintillation liquid to the filter plates(Perkin Elmer).

When Compound 1 was tested in this assay, a Ki value of 6.9 nM wasmeasured.

Alternatively, for the determination of Ki, different amounts ofinhibitor were mixed with the enzyme and the enzymatic reaction wasfollowed as a function of ATP concentration. The Ki was determined bymeans of double reciprocal plotting of Km vs compound concentration(Lineweaver-Burk plot). 1 ng of JAK1 (Invitrogen, PV4774) was used inthe assay. The substrate was 50 nM Ulight-JAK-1 (Tyr1023) Peptide(Perkin Elmer, TRF0121) The reaction was performed in 25 mM MOPS pH 6.8,0.01%, 2 mM DTT, 5 mM MgCl₂ Brij-35 with varying concentrations of ATPand compound. Phosphorylated substrate was measured using an Eu-labeledanti-phosphotyrosine antibody PT66 (Perkin Elmer, AD0068). Readout wasperformed on the envision (Perkin Elmer) with excitation at 320 nm andemission followed at 615 nm and 665 nm.

When Compound 1 was tested in this assay, a Ki value of 5.6 nM wasmeasured.

Example 1.3 JAK2 Inhibition Assay

Recombinant human JAK2 (catalytic domain, amino acids 866-1154; catalognumber PV4210) was purchased from Invitrogen. 0.0125 mU of JAK2 wasincubated with 25 nM Ulight-JAK1(tyr1023) peptide (Perkin Elmer catalognumber TRF0121) in kinase reaction buffer (41.66 mM HEPES pH7.0, 0.016%Triton X-100, 12.5 mM MgCl₂, 3.33 mM DTT, 7.5 μM ATP) with or without 4μL containing test compound or vehicle (DMSO, 1% final concentration),in a total volume of 20 μL, in a white 384 Luminotrac 200 plate(Greiner, catalog number 781075). After 60 min at room temperature,reactions were stopped by adding 20 μL/well of detection mixture (1×detection buffer (Perkin Elmer, catalog number CR97-100C), 0.5 nMEuropium-anti-phosphotyrosine (PT66) (Perkin Elmer, catalog numberAD0068), 10 mM EDTA). Readout is performed using the Envision withexcitation at 320 nm and measuring emission at 615 nm (Perkin Elmer).Kinase activity was calculated by subtracting relative fluorescenceunits (RFU) obtained in the presence of a positive control inhibitor (10μM staurosporine) from RFU obtained in the presence of vehicle. Theability of a test compound to inhibit this activity was determined as:

Percentage inhibition=((RFU determined for sample with test compoundpresent−RFU determined for sample with positive control inhibitor)divided by (RFU determined in the presence of vehicle−RFU determined forsample with positive control inhibitor))*100.

Dose dilution series are prepared for compound enabling the testing ofdose-response effects in the JAK2 assay and the calculation of the IC₅₀for the compound. Each compound is routinely tested at concentration of20 μM followed by a 1/5 serial dilution, 8 points (20 μM-4 μM-800 nM-160nM-32 nM-6.4 nM-1.28 nM-0.26 nM) in a final concentration of 1% DMSO.When potency of compound series increases, more dilutions are preparedand/or the top concentration are lowered (e.g. 5 μM, 1 μM). The data areexpressed as the average IC₅₀ from the assays±standard error of themean.

TABLE IV JAK2 IC₅₀ Values of Compound Cpd # JAK2 (nM) 1 67 ± 5

Example 1.4 JAK2 Ki Determination Assay

For the determination of Ki, different amounts of inhibitor were mixedwith the enzyme and the enzymatic reaction was followed as a function ofATP concentration. The Ki was determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). 0.025mU of JAK2 (Invitrogen, PV4210) was used in the assay. The substrate wasPoly(Glu,Tyr)sodium salt (4:1), MW 20 000-50 000 (Sigma, Pb0275) Thereaction was performed in 10 mM MOPS pH 7.5, 0.5 mM EDTA, 0.01% Brij-35,1 mM DTT, 15 mM MgAc with varying concentrations of ATP and compound andstopped by addition of 150 mM phosphoric acid. Measurement ofincorporated phosphate into the substrate polyGT was done by loading thesamples on a filter plate (using a harvester, Perkin Elmer) andsubsequent washing. Incorporated ³³P in polyGT is measured in a Topcountscintillation counter after addition of scintillation liquid to thefilter plates (Perkin Elmer).

When Compound 1 was tested in this assay, a Ki value of 126 nM wasmeasured.

Aternatively, for the determination of Ki, different amounts ofinhibitor were mixed with the enzyme and the enzymatic reaction wasfollowed as a function of ATP concentration. The Ki was determined bymeans of double reciprocal plotting of Km vs compound concentration(Lineweaver-Burk plot). 0.0125 mU of JAK2 (Invitrogen, PV4210) was usedin the assay. The substrate was 50 nM Ulight-JAK-1 (Tyr1023) Peptide(Perkin Elmer, TRF0121). The reaction was performed in 25 mM HEPES pH7.0, 0.01% Triton X-100, 2 mM DTT, 7.5 mM MgCl₂ with varyingconcentrations of ATP and compound. Phosphorylated substrate wasmeasured using an Eu-labeled anti-phosphotyrosine antibody PT66 (PerkinElmer, AD0068). Readout was performed on the envision (Perkin Elmer)with excitation at 320 nm and emission followed at 615 nm and 665 nm.

When Compound 1 was tested in this assay, a Ki value of 35 nM wasmeasured.

Example 1.5 JAK3 Inhibition Assay

Recombinant human JAK3 catalytic domain (amino acids 781-1124; catalognumber PV3855) was purchased from Invitrogen. 0.025 mU of JAK3 wasincubated with 2.5 μg polyGT substrate (Sigma catalog number Pb0275) inkinase reaction buffer (25 mM Tris pH 7.5, 0.5 mM EGTA, 0.5 mM Na₃VO₄, 5mM b-glycerolphosphate, 0.01% Triton X-100, 1 μM non-radioactive ATP,0.25 μCi ³³P-gamma-ATP (GE Healthcare, catalog number AH9968) finalconcentrations) with or without 5 μL containing test compound or vehicle(DMSO, 1% final concentration), in a total volume of 25 μL, in apolypropylene 96-well plate (Greiner, V-bottom). After 105 min at 30°C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoricacid. All of the terminated kinase reaction was transferred to prewashed(75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalognumber 6005177) using a cell harvester (Perkin Elmer). Plates werewashed 6 times with 300 μL per well of a 75 mM phosphoric acid solutionand the bottom of the plates was sealed. 40 μL/well of Microscint-20 wasadded, the top of the plates was sealed and readout was performed usingthe Topcount (Perkin Elmer). Kinase activity was calculated bysubtracting counts per minute (cpm) obtained in the presence of apositive control inhibitor (10 μM staurosporine) from cpm obtained inthe presence of vehicle. The ability of a test compound to inhibit thisactivity was determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compound enabling the testingof dose-response effects in the JAK3 assay and the calculation of theIC₅₀ for the compound. Each compound was routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of the compound series increased,more dilutions were prepared and/or the top concentration was lowered(e.g. 5 μM, 1 μM). The data are expressed as the average IC₅₀ from theassays±standard error of the mean.

TABLE V JAK3 IC₅₀ Values of Compound Cpd # JAK3 (nM) 1 408 ± 62

Example 1.6 JAK3 Ki Determination Assay

For the determination of Ki, different amounts of inhibitor were mixedwith the enzyme and the enzymatic reaction was followed as a function ofATP concentration. The Ki was determined by means of double reciprocalplotting of Km vs compound concentration (Lineweaver-Burk plot). JAK3(Carna Biosciences, 09CBS-0625B) was used at a final concentration of 10ng/ml. The substrate was Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50000 (Sigma, Pb0275) The reaction was performed in 25 mM Tris pH 7.5,0.01% Triton X-100, 0.5 mM EGTA, 2.5 mM DTT, 0.5 mM Na₃VO₄, 5 mMb-glycerolphosphate, 10 mM MgCl₂ with varying concentrations of ATP andcompound and stopped by addition of 150 mM phosphoric acid. Measurementof incorporated phosphate into the substrate polyGT was done by loadingthe samples on a filter plate (using a harvester, Perkin Elmer) andsubsequent washing. Incorporated ³³P in polyGT is measured in a Topcountscintillation counter after addition of scintillation liquid to thefilter plates (Perkin Elmer)

When Compound 1 was tested in this assay, a Ki value of 188 nM wasmeasured.

Example 1.7 TYK2 Inhibition Assay

Recombinant human TYK2 catalytic domain (amino acids 871-1187; catalognumber 08-147) was purchased from Carna biosciences. 5 ng of TYK2 wasincubated with 12.5 μg polyGT substrate (Sigma catalog number Pb0275) inkinase reaction buffer (25 mM Hepes pH 7.5, 100 mM NaCl, 0.2 mM Na₃VO₄,0.1% NP-40, 0.1 μM non-radioactive ATP, 0.125 μCi ³³P-gamma-ATP (GEHealthcare, catalog number AH9968) final concentrations) with or without5 μL containing test compound or vehicle (DMSO, 1% final concentration),in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner,V-bottom). After 90 min at 30° C., reactions were stopped by adding of25 μL/well of 150 mM phosphoric acid. All of the terminated kinasereaction was transferred to prewashed (75 mM phosphoric acid) 96 wellfilter plates (Perkin Elmer catalog number 6005177) using a cellharvester (Perkin Elmer). Plates were washed 6 times with 300 μL perwell of a 75 mM phosphoric acid solution and the bottom of the plateswas sealed. 40 μL/well of Microscint-20 was added, the top of the plateswas sealed and readout was performed using the Topcount (Perkin Elmer).Kinase activity was calculated by subtracting counts per minute (cpm)obtained in the presence of a positive control inhibitor (10 μMstaurosporine) from cpm obtained in the presence of vehicle. The abilityof a test compound to inhibit this activity was determined as:

Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100.

Dose dilution series were prepared for the compounds enabling thetesting of dose-response effects in the TYK2 assay and the calculationof the IC₅₀ for the compound. Each compound was routinely tested atconcentration of 20 μM followed by a 1/3 serial dilution, 8 points (20μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. When potency of compound series increased,more dilutions were prepared and/or the top concentration was lowered(e.g. 5 μM, 1 μM).

TABLE VI TYK2 IC₅₀ Values of Compound Cpd # TYK2 (nM) 1 219 ± 37

Example 2 Cellular Assays Example 2.1 JAK-STAT Signalling Assay

HeLa cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)containing 10% heat inactivated fetal calf serum, 100 U/mL penicillinand 100 μg/mL streptomycin. HeLa cells were used at 70% confluence fortransfection. 20,000 cells in 87 μL cell culture medium were transientlytransfected with 40 ng pSTAT1(2)-luciferase reporter (Panomics), 8 ng ofLacZ reporter as internal control reporter and 52 ng of pBSK using 0.32μL Jet-PEI (Polyplus) as transfection reagent per well in 96-well plateformat. After overnight incubation at 37° C., 10% CO₂, transfectionmedium was removed. 75 μL of DMEM+1.5% heat inactivated fetal calf serumwas added. 15 μL of compound at 6.7× concentration was added for 60 minand then 10 μL of human OSM (Peprotech) at 33 ng/mL final concentration.

All compounds were tested in duplicate starting from 20 μM followed by a1/3 serial dilution, 8 doses in total (20 μM-6.6 μM-2.2 μM-740 nM-250nM-82 nM-27 nM-9 nM) in a final concentration of 0.2% DMSO.

After overnight incubation at 37° C., 10% CO₂ cells were lysed in 100 μLlysis buffer/well (PBS, 0.9 mM CaCl₂, 0.5 mM MgCl₂, 5% Trehalose, 0.025%Tergitol NP9, 0.15% BSA).

40 μL of cell lysate was used to read β-galactosidase activity by adding180 μL βGal solution (30 μl ONPG 4 mg/mL+150 μL β-Galactosidase buffer(0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄, 1 mM MgCl₂)) for 20 min. The reactionwas stopped by addition of 50 μL Na₂CO₃ 1 M. Absorbance was read at 405nm.

Luciferase activity was measured using 40 μL cell lysate plus 40 μl ofSteadylite® as described by the manufacturer (Perkin Elmer), on theEnvision (Perkin Elmer).

10 μM of a pan-JAK inhibitor was used as a positive control (100%inhibition). As negative control 0.5% DMSO (0% inhibition) was used. Thepositive and negative controls were used to calculate z′ and ‘percentinhibition’ (PIN) values.

Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100.

PIN values were plotted for compounds tested in dose-response and EC₅₀values were derived.

TABLE VII STAT signalling EC₅₀ Values of Compound Cpd # EC₅₀ (nM) 1 539± 130

Example 2.2 OSM/IL-1β signaling Assay

OSM and IL-1β were shown to synergistically upregulate MMP13 levels inthe human chondrosarcoma cell line SW1353. The cells were seeded in 96well plates at 15,000 cells/well in a volume of 120 μL DMEM (Invitrogen)containing 10% (v/v) FBS and 1% penicillin/streptomycin (InVitrogen)incubated at 37° C. 5% CO₂. Cells were preincubated with 15 μL compoundin M199 medium with 2% DMSO 1 hr before triggering with 15 μL OSM andIL-1β to reach 25 ng/mL OSM and 1 ng/mL IL-1β, and MMP13 levels weremeasured in conditioned medium 48 hours after triggering. MMP13 activitywas measured using an antibody capture activity assay. For this purpose,384 well plates (NUNC, 460518, MaxiSorb black) were coated with 35 μL ofa 1.5 μg/mL anti-human MMP13 antibody (R&D Systems, MAB511) solution for24 hours at 4° C. After washing the wells 2 times with PBS+0.05% Tween,the remaining binding sites were blocked with 100 μL 5% non-fat dry milk(Santa Cruz, sc-2325, Blotto) in PBS for 24 hours at 4° C. Next, thewells were washed 2 times with PBS+0.05% Tween and 35 μL of 1/10dilution of culture supernatant containing MMP13 in 100-fold dilutedblocking buffer was added and incubated for 4 hours at room temperature.Next the wells were washed twice with PBS+0.05% Tween followed by MMP13activation by addition of 35 μL of a 1.5 mM 4-Aminophenylmercuricacetate (APMA) (Sigma, A9563) solution and incubation at 37° C. for 1hour. The wells were washed again with PBS+0.05% Tween and 35 μL MMP13substrate (Biomol, P-126, OmniMMP fluorogenic substrate) was added.After incubation for 24 hours at 37° C. fluorescence of the convertedsubstrate was measured in a Perkin Elmer Wallac EnVision 2102 MultilabelReader (wavelength excitation: 320 nm, wavelength emission: 405 nm).

Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100.

The data are expressed as the average EC₅₀ from the assays±standarderror of the mean.

TABLE VIII MMP13 EC₅₀ Values of Compounds Cpd # EC₅₀ (nM) 1 4196 ± 2370

Example 2.3 PBL Proliferation assay

Human peripheral blood lymphocytes (PBL) are stimulated with IL-2 andproliferation measured using a BrdU incorporation assay. The PBL arefirst stimulated for 72 hrs with PHA to induce IL-2 receptor, fasted for24 hrs to stop cell proliferation followed by IL-2 stimulation foranother 72 hrs (including 24 hr BrdU labeling). Cells are preincubatedwith test compounds 1 hr before IL-2 addition. Cells are cultured inRPMI 1640 containing 10% (v/v) FBS.

Example 2.4 Whole Blood Assays (WBA) 2.4.1 IFNα Stimulation Protocol

To predict the selectivity of the test compounds to inhibit JAK1- overJAK2-dependent signaling pathways in vivo, a physiologically relevant invitro model was developed using human whole blood. In the WBA assay,blood drawn from human volunteers who gave informed consent, was treatedex vivo with compound (1 h) and subsequently stimulated either for 30minutes with interferon α (IFNα, JAK1 dependent pathway) or for 2 h withgranulocyte macrophage-colony stimulating factor (GM-CSF, JAK2 dependentpathway).

2.4.1.1 Phospho-STAT1 Assay

For IFNα stimulation, increase in phosphorylation of Signal Transducersand Activators of Transcription 1 (pSTAT1) by IFNα in white blood cellextracts was measured using a pSTAT1 ELISA assay. Phosphorylation ofSignal Transducer and Activator of Transcription 1 (STAT1) afterinterferon alpha (IFNα) triggering is a JAK1-mediated event. ThePhospho-STAT1 Assay, which was used to measure Phospho-STAT1 levels incellular extracts, was developed to assess the ability of a compound toinhibit JAK1-dependent signaling pathways.

Whole human blood, drawn from human volunteers who gave informedconsent, was ex vivo treated with compound (1 h) and subsequentlystimulated for 30 minutes with IFNα. The increase in phosphorylation ofSTAT1 by IFNα in white blood cell extracts was measured using aphospho-STAT1 ELISA.

The ACK lysis buffer consisted of 0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mMEDTA. The pH of the buffer was 7.3.

A 10× cell lysis buffer concentrate (part of the PathScan Phospho-STAT1(Tyr701) sandwich ELISA kit from Cell Signaling) was diluted 10-fold inH₂O. Proteinase inhibitors were added to the buffer before use.

20 μg IFNα is dissolved in 40 μL H₂O to obtain a 500 μg/mL stocksolution. The stock solution was stored at −20° C.

A 3-fold dilution series of the compound was prepared in DMSO (highestconcentration: 10 mM). Subsequently, the compound was further diluted inmedium (dilution factor dependent on desired final compoundconcentration).

2.4.1.1.1 Incubation of Blood with Compound and Stimulation with IFNα

Human blood was collected in heparinized tubes. The blood was divided inaliquots of 392 μL. Afterwards, 4 μL of compound dilution was added toeach aliquot and the blood samples were incubated for 1 h at 37° C. TheIFNα stock solution was diluted 1000-fold in RPMI medium to obtain a 500ng/mL working solution. 4 μL of the 500 ng/mL work solution was added tothe blood samples (final concentration IFNα: 5 ng/ml). The samples wereincubated at 37° C. for 30 min.

2.4.1.1.2 Preparation of Cell Extracts

At the end of the stimulation period, 7.6 mL ACK buffer was added to theblood samples to lyse the red blood cells. The samples were mixed byinverting the tubes five times and the reaction was incubated on ice for5 min. The lysis of the RBC should be evident during this incubation.The cells were pelleted by centrifugation at 300 g, 4° C. for 7 min andthe supernatant was removed. 10 mL 1×PBS was added to each tube and thecell pellet was resuspended. The samples were centrifuged again for 7min at 300 g, 4° C. The supernatant was removed and the pelletresuspended in 500 μL of 1×PBS. Then, the cell suspension wastransferred to a clean 1.5 mL microcentrifuge tube. The cells werepelleted by centrifugation at 700 g for 5 min at 4° C. The supernatantwas removed and the pellet was dissolved in 150 μL cell lysis buffer.The samples were incubated on ice for 15 min. After that, the sampleswere stored at −80° C. until further processing.

2.4.1.1.3 Measurement of STAT1 Phosphorylation by ELISA

The Pathscan Phospho-STAT1 (Tyr701) Sandwich ELISA kit from CellSignaling (Cat. no: #7234) was used to determine Phospho-STAT1 levels.

The cellular extracts were thawed on ice. The tubes were centrifuged for5 min at 16,000 g, 4° C. and the cleared lysates were harvested.Meanwhile, the microwell strips from the kit were equilibrated to roomtemperature and wash buffer was prepared by diluting 20× wash buffer inH₂0. Samples were diluted 2-fold in sample diluent and 100 μL was addedto the microwell strips. The strips were incubated overnight at 4° C.

The following day, the wells were washed 3 times with wash buffer. 100μL of the detection antibody was added to the wells. The strips wereincubated at 37° C. for 1 h. Then, the wells were washed 3 times withwash buffer again. 100 μL HRP-linked secondary antibody was added toeach well and the samples were incubated at 37° C. After 30 min, thewells were washed 3 times again and 100 μL TMB substrate was added toall wells. When samples turned blue, 100 μL STOP solution was added tostop the reaction. Absorbance was measured at 450 nm.

2.4.1.2 IL-8 ELISA

For GM-CSF stimulation, increase in interleukin-8 (IL-8) levels inplasma was measured using an IL-8 ELISA assay. Granulocytemacrophage-colony stimulating factor (GM-CSF)-induced interleukin 8(IL-8) expression is a JAK2-mediated event. The IL-8 ELISA, which can beused to measure IL-8 levels in plasma samples, was developed to assessthe ability of a compound to inhibit JAK2-dependent signaling pathways.

Whole human blood, drawn from human volunteers who gave informedconsent, was ex vivo treated with compound (1 h) and subsequentlystimulated for 2 h with GM-CSF. The increase in IL-8 levels in plasmawas measured using an IL-8 ELISA assay.

10 μg GM-CSF was dissolved in 100 μL H₂O to obtain a 100 μg/mL stocksolution. The stock solution was stored at −20° C.

A 3-fold dilution series of the test compound was prepared in DMSO(highest concentration: 10 mM). Subsequently, the compound was furtherdiluted in medium (dilution factor dependent on desired final compoundconcentration).

2.4.1.2.1 Incubation of Blood with Compound and Stimulation with GM-CSF

Human blood was collected in heparinized tubes. The blood was divided inaliquots of 245 μL. Afterwards, 2.5 μL test compound dilution was addedto each aliquot and the blood samples were incubated for 1 h at 37° C.The GM-CSF stock solution was diluted 100-fold in RPMI medium to obtaina 1 μg/mL work solution. 2.5 μL of the 1 μg/mL work solution was addedto the blood samples (final concentration GM-CSF: 10 ng/mL). The sampleswere incubated at 37° C. for 2 h.

2.4.1.2.2 Preparation of Plasma Samples

The samples were centrifuged for 15 min at 1,000 g, 4° C. 100 μL of theplasma was harvested and stored at −80° C. until further use.

2.4.1.2.3 Measurement of IL-8 levels by ELISA

The Human IL-8 Chemiluminescent Immunoassay kit from R&D Systems (Cat.no: Q8000B) was used to determine IL-8 levels.

Wash buffer was prepared by diluting 10× wash buffer in H₂O. Working gloreagent was prepared by adding 1 part Glo Reagent 1 to 2 parts GloReagent B 15 min to 4 h before use.

100 μL assay diluent RD1-86 was added to each well. After that, 50 μL ofsample (plasma) was added. The ELISA plate was incubated for 2 h at roomtemperature, 500 rpm. All wells were washed 4 times with wash buffer and200 μL IL-8 conjugate was added to each well. After incubation for 3 hat room temperature, the wells were washed 4 times with wash buffer and100 μL working glo reagent was added to each well. The ELISA plate wasincubated for 5 min at room temperature (protected from light).Luminescence was measured (0.5 s/well read time).

2.4.1.3 Results

The pIC₅₀ of Compound 1 for inhibiting the INFα induced increase ofpSTAT1 levels was 6.32±0.07 (SEM), while pIC₅₀ for inhibition of GM-CSFinduced increase of IL-8 was 4.87±0.13 (SEM) (results obtained using 6blood donors). This demonstrates that Compound 1 is 28-fold moreselective for the JAK1 pathway versus the JAK2 pathway. Therefore, in acellular setting it is clear that Compound 1 exhibits selectivity forJAK1 over JAK2. Compound 1 seems to exhibit a greater selectivity forJAK1 over JAK2 compared to known structurally related compounds, orother known JAK inhibitors.

2.4.1.3 Data Curation Treatment and Results

In order to further refine the results, inhibition of phosphoSTAT1induction by IFNα in cell extracts or inhibition of IL-8 induction byGM-CSF in plasma were plotted against the compound concentration andIC₅₀ values were derived using Graphpad software. Data were retained ifR² was larger than 0.8 and the hill slope was smaller than 3 and onlydonors for which valid data were obtained for both assays were retained.

For example, when submitted to this additional data analysis protocol,the measured pIC₅₀ of Compound 1 for inhibiting the INFα inducedincrease of pSTAT1 levels was 6.21±0.09 (SEM), while pIC₅₀ forinhibition of GM-CSF induced increase of IL-8 was 4.97±0.14 (SEM)(results obtained using 6 blood donors). This confirms that Compound 1is 17.6-fold more selective for the JAK1 pathway versus the JAK2pathway. Therefore, in a cellular setting it is clear that Compound 1exhibits selectivity for JAK1 over JAK2. Compound 1 seems to exhibit agreater selectivity for JAK1 over JAK2 compared to known structurallyrelated compounds, or other known JAK inhibitors.

2.4.2 IL-6 Stimulation Protocol

A flow cytometry analysis was also performed to establish compoundselectivity for JAK1 over JAK2, ex vivo using human whole blood.Therefore, blood was taken from human volunteers who gave informedconsent. Blood was then equilibrated for 30 minutes at 37° C. undergentle rocking, then aliquoted in Eppendorf tubes. Compound was added atdifferent concentrations and incubated at 37° C. for 30 minutes undergentle rocking and subsequently stimulated for 20 minutes at 37° C.under gentle rocking with interleukin 6 (IL-6) for JAK1-dependentpathway stimulation or GM-CSF for JAK2-dependent pathway stimulation.Phospho-STAT1 and phospho-STAT5 were then evaluated using FACS analysis.

2.4.2.1 Phospho-STAT1 Assays

For IL-6-stimulated increase of Signal Transducers and Activators ofTranscription 1 (pSTAT1) phosphorylation in white blood cell, humanwhole blood, drawn from human volunteers who gave informed consent, wasex vivo treated with the compound for 30 min and subsequently stimulatedfor 20 minutes with IL-6. The increase in phosphorylation of STAT1 byIL-6 in lymphocytes was measured using anti phospho-STAT1 antibody byFACS.

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. No. 558049) was diluted 5-foldwith distilled water and pre-warmed at 37° C. Remaining diluted Lyse/Fixbuffer was discarded.

10 μg rhIL-6 (R&D Systems, Cat No. 206-IL) was dissolved in 1 ml of PBS0.1% BSA to obtain a 10 μg/ml stock solution. The stock solution wasstored aliquoted at −80° C.

A 3-fold dilution series of the compound was prepared in DMSO (10 mMstock solution). Control-treated samples received DMSO instead ofcompound. All samples were incubated with a 1% final DMSO concentration.

2.4.2.1.1 Incubation of Blood with Compound and Stimulation with IL-6

Human blood was collected in heparinized tubes. The blood was divided inaliquots of 148.54 μl. Then, 1.5 μl of compound dilution was added toeach aliquot and the blood samples were incubated for 30 min at 37° C.under gentle rocking. IL-6 stock solution (1.5 μl) was added to theblood samples (final concentration 10 ng/ml) and samples were incubatedat 37° C. for 20 min under gentle rocking.

2.4.2.1.2 White Blood Cell Preparation and CD4 Labeling

At the end of the stimulation period, 3 ml of 1× pre-warmed Lyse/Fixbuffer was immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes, then frozen at −80° C. until furtheruse.

For the following steps, tubes were thawed at 37° C. for approximately20 minutes and centrifuged for 5 min at 400 g at 4° C. The cell pelletwas washed with 3 ml of cold 1×PBS, and after centrifugation the cellpellet was resuspended in 100 μl of PBS containing 3% BSA.FITC-conjugated anti-CD4 antibody or control FITC-conjugated isotypeantibody were added and incubated for 20 min at room temperature, in thedark.

2.4.2.1.3 Cell Permeabilization and Labeling with Anti Phospho-STAT1Antibody

After washing cells with 1×PBS, the cell pellet was resuspended in 100μl of ice-cold 1×PBS and 900 μl of ice-cold 100% methanol was added.Cells were then incubated at 4° C. for 30 min for permeabilization.

Permeabilized cells were then washed with 1×PBS containing 3% BSA andfinally resuspended in 80 μl of 1×PBX containing 3% BSA.

20 μL of PE mouse anti-STAT1 (pY701) or PE mouse IgG2aκ isotype controlantibody (BD Biosciences, Cat. No. 612564 and 559319, respectively) wereadded and mixed, then incubated for 30 min at 4° C., in the dark.

Cells are then washed once with 1×PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

2.4.2.1.4 Fluorescence Analysis on FACSCanto 11

50,000 total events were counted and Phospho-STAT1 positive cells weremeasured after gating on CD4+ cells, in the lymphocyte gate. Data wereanalyzed using the FACSDiva software and correspond to the percentage ofinhibition of IL-6 stimulation calculated on the percentage of positivecells for phospho-STAT1 on CD4+ cells.

2.4.2.2 Phospho-STAT5 Assay

For GM-CSF-stimulated increase of Signal Transducers and Activators ofTranscription 5 (pSTAT5) phosphorylation in white blood cell, humanwhole blood, drawn from human volunteers who gave informed consent, wasex vivo treated with compound for 30 min and subsequently stimulated for20 minutes with GM-CSF. The increase in phosphorylation of STAT5 byGM-CSF in monocytes was measured using an anti phospho-STAT5 antibody byFACS.

The 5× Lyse/Fix buffer (BD PhosFlow, Cat. No. 558049) was diluted 5-foldwith distilled water and pre-warmed at 37° C. Remaining diluted Lyse/Fixbuffer was discarded.

10 μg rhGM-CSF (AbCys S.A., Cat No. P300-03) was dissolved in 100 μl ofPBS 0.1% BSA to obtain a 100 μg/ml stock solution. The stock solutionwas stored aliquoted at −80° C.

A 3-fold dilution series of the compound was prepared in DMSO (10 mMstock solution). Control-treated samples received DMSO without any testcompound. All samples were incubated with a 1% final DMSO concentration.

2.4.2.2.1 Incubation of Blood with Compound and Stimulation with GM-CSF

Human blood was collected in heparinized tubes. The blood was divided inaliquots of 148.5 μl. Afterwards, 1.5 μl of compound dilution was addedto each aliquot and the blood samples were incubated for 30 min at 37°C. under gentle rocking. GM-CSF stock solution (1.5 μl) was added to theblood samples (final concentration 20 pg/ml) and samples were incubatedat 37° C. for 20 min under gentle rocking.

2.4.2.2.2 White Blood Cell Preparation and CD14 Labeling

At the end of the stimulation period, 3 ml of 1× pre-warmed Lyse/Fixbuffer was immediately added to the blood samples, vortexed briefly andincubated for 15 min at 37° C. in a water bath in order to lyse redblood cells and fix leukocytes, then frozen at −80° C. until furtheruse.

For the following steps, tubes were thawed at 37° C. for approximately20 minutes and centrifuged for 5 min at 400 g at 4° C. The cell pelletwas washed with 3 ml of cold 1×PBS, and after centrifugation the cellpellet was resuspended in 100 μl of PBS containing 3% BSA. FITC mouseanti-CD14 antibody (BD Biosciences, Cat. No. 345784) or control FITCmouse IgG2bκ isotype antibody (BD Biosciences, Cat. No. 555057) wereadded and incubated for 20 min at room temperature, in the dark.

2.4.2.2.3 Cell Permeabilization and Labeling with Anti Phospho-STAT5Antibody

After washing cells with 1×PBS, the cell pellet was resuspended in 100μl of ice-cold 1×PBS and 900 μl of ice-cold 100% methanol was added.Cells were then incubated at 4° C. for 30 min for permeabilization.

Permeabilized cells were then washed with 1×PBS containing 3% BSA andfinally resuspended in 80 μl of 1×PBS containing 3% BSA.

20 μL of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype controlantibody (BD Biosciences, Cat. No. 612567 and 554680, respectively) wereadded and mixed then incubated for 30 min at 4° C., in the dark.

Cells are then washed once with 1×PBS and analyzed on a FACSCanto IIflow cytometer (BD Biosciences).

2.4.2.2.4 Fluorescence Analysis on FACSCanto II

50,000 total events were counted and Phospho-STAT5 positive cells weremeasured after gating on CD14+ cells. Data were analyzed using theFACSDiva software and correspond to the percentage of inhibition ofGM-CSF stimulation calculated on the percentage of positive cells forphospho-STAT5 on CD14+ cells.

2.4.2.3 Results

When submitted to this protocol, the perrcentage of inhibition (PIN)obtained from the mean of 3 healthy volunteers was determined for thecompound of the invention. For example, Compound 1 was tested andreturned a pIC₅₀=6.61 in the inhibition of STAT1 phosphorylation and apIC₅₀=5.37 in the inhibition of STAT5 phosphorylation

When comparing the effect of Compound 1 on STAT1 (JAK1-dependentpathway) and STAT5 (JAK2-dependent pathway), a 17.6-fold selectivity forJAK1 vs. JAK2 was measured.

Example 3 In Vivo Models Example 3.1 CIA Model 3.1.1 Materials

Completed Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)were purchased from Difco. Bovine collagen type TT (CII),lipopolysaccharide (LPS), and Enbrel were obtained from Chondrex (Isled'Abeau, France); Sigma (P4252, L'Isle d'Abeau, France), Whyett (25 mginjectable syringe, France) Acros Organics (Palo Alto, Calif.),respectively. All other reagents used were of reagent grade and allsolvents were of analytical grade.

3.1.2 Animals

Dark Agouti rats (male, 7-8 weeks old) are obtained from HarlanLaboratories (Maison-Alfort, France). DBA/1J mice (male, 7 weeks old)were obtained from Centre d'Elevage et de Reproduction JANVIER (CERJ)(Laval, France). Rats and mice were kept on a 12 hours light/dark cycle(0700-1900). The temperature was maintained at 22° C., and food andwater were provided ad libitum.

3.1.3 Collagen Induced Arthritis (CIA)

One day before the experiment, CII solution (2 mg/mL) was prepared with0.05 M acetic acid and stored at 4° C. Just before the immunization,equal volumes of adjuvant (IFA) and CII were mixed by a homogenizer in apre-cooled glass bottle in an ice water bath. Extra adjuvant andprolonged homogenization might be required if an emulsion is not formed.

Mice: 0.1 mL of the emulsion was injected intradermally at the base ofthe tail of each mouse on day 1, a second booster intradermal injection(CII solution at 1 mg/mL in CFA 0.1 mL saline) was performed on day 21.This immunization method was modified from published methods (David DBrand Kary A Latham, & Edward F Rosloniec. Collagen-induced arthritis.Nature Methods 2 (5): 1269-1275, 2007).

Rat: 0.2 mL of the emulsion is injected intradermally at the base of thetail of each rat on day 1, a second booster intradermal injection (CIIsolution at 2 mg/mL in CFA 0.1 mL saline) is performed on day 9. Thisimmunization method is modified from published methods (Sims N A et al.,(2004) Targeting osteoclasts with zoledronic acid prevents bonedestruction in collagen-induced arthritis, Arthritis Rheum. 502338-2346; Jou et al., 2005).

3.1.4 Study Design

The therapeutic effects of the test compounds were tested in the rat ormouse CIA model. Animals were randomly divided into equal groups andeach group contained 10 animals. All rats are immunized on day 1 andboosted on day 9. All mice were immunized on day 1 and boosted on day21. Therapeutic dosing lasted from day 16 to day 30. The negativecontrol group was treated with vehicle (MC 0.5%) and the positivecontrol group with Enbrel (10 mg/kg, 3× week., s.c.). A compound ofinterest was typically tested at 3 doses, e.g. 3, 10, 30 mg/kg, p.o.

3.1.5 Clinical Assessment of Arthritis

Arthritis was scored according the method of Khachigian 2006, Lin et al2007 and Nishida et al. 2004). The swelling of each of the four paws wasranked with the arthritic score as follows: 0-no symptoms; 1-mild, butdefinite redness and swelling of one type of joint such as the ankle orwrist, or apparent redness and swelling limited to individual digits,regardless of the number of affected digits; 2-moderate redness andswelling of two or more types of joints; 3-severe redness and swellingof the entire paw including digits; 4-maximally inflamed limb withinvolvement of multiple joints (maximum cumulative clinical arthritisscore 16 per animal) (Nishida et al., 2004).

To permit the meta-analysis of multiple studies the clinical scorevalues were normalised as follows:

AUC of Clinical Score (AUC Score):

The area under the curve (AUC) from day 1 to day 14 was calculated foreach individual rat. The AUC of each animal was divided by the averageAUC obtained for the vehicle in the study from which the data on thatanimal was obtained and multiplied by 100 (i.e. the AUC was expressed asa percentage of the average vehicle AUC per study).

Clinical Score Increase from Day 1 to Day 14 (End Point Score):

The clinical score difference for each animal was divided by the averageclinical score difference obtained for the vehicle in the study fromwhich the data on that animal was obtained and multiplied by 100 (i.e.the difference was expressed as a percentage of the average clinicalscore difference for the vehicle per study).

3.1.6 Change in Body Weight (%) after Onset of Arthritis

Clinically, body weight loss is associated with arthritis (Shelton etal., 2005; Argiles et al., 1998; Rall, 2004; Walsmith et al., 2004).Hence, changes in body weight after onset of arthritis could be used asa non-specific endpoint to evaluate the effect of therapeutics in therat model. The change in body weight (%) after onset of arthritis wascalculated as follows:

${Mice}\text{:}\mspace{14mu} \frac{{{Body}\mspace{14mu} {Weight}_{({{week}\; 6})}} - {{Body}\mspace{14mu} {Weight}_{({{week}\; 5})}}}{{Body}\mspace{14mu} {Weight}_{({{week}\; 5})}} \times 100\%$${Rats}\text{:}\mspace{14mu} \frac{{{Body}\mspace{14mu} {Weight}_{({{week}\; 4})}} - {{Body}\mspace{14mu} {Weight}_{({{week}\; 3})}}}{{Body}\mspace{14mu} {Weight}_{({{week}\; 3})}} \times 100\%$

3.1.7 Larsen’ Score

Post-mortem, a Larsen’ score was derived for both hind paws of all ratsby at least 2 scientists. The average of these scores was calculated toobtain one Larsen’ score per rat. To allow an inter-study comparison ofthe Larsen’ scores, the Larsen’ score of each rat was divided by theaverage Larsen’ score obtained for vehicle in the study to which thatrat belongs and multiplied by 100 (i.e. expressing the Larsen’ score asthe percentage of average vehicle Larsen’ score per study). The averageLarsen’ score of the different treatment groups was calculated andcompared.

3.1.8 Radiology

X-ray photos were taken of the hind paws of each individual animal. Arandom blind identity number was assigned to each of the photos, and theseverity of bone erosion was ranked by two independent scorers with theradiological Larsen’s score system as follows: 0-normal with intact bonyoutlines and normal joint space; 1-slight abnormality with any one ortwo of the exterior metatarsal bones showing slight bone erosion;2-definite early abnormality with any three to five of the exteriormetatarsal bones showing bone erosion; 3-medium destructive abnormalitywith all the exterior metatarsal bones as well as any one or two of theinterior metatarsal bones showing definite bone erosions; 4-severedestructive abnormality with all the metatarsal bones showing definitebone erosion and at least one of the inner metatarsal joints completelyeroded leaving some bony joint outlines partly preserved; 5-mutilatingabnormality without bony outlines. This scoring system is a modificationfrom Salvemini et al., 2001; Bush et al., 2002; Sims et al., 2004; Jouet al., 2005.

3.1.9 Histology

After radiological analysis, the hind paws of mice were fixed in 10%phosphate-buffered formalin (pH 7.4), decalcified with rapid bonedecalcificant for fine histology (Laboratories Eurobio) and embedded inparaffin. To ensure extensive evaluation of the arthritic joints, atleast four serial sections (5 μm thick) were cut and each series ofsections were 100 μm in between. The sections were stained withhematoxylin and eosin (H&E). Histologic examinations for synovialinflammation and bone and cartilage damage were performed double blind.In each paw, four parameters were assessed using a four-point scale. Theparameters were cell infiltration, pannus severity, cartilage erosionand bone erosion. Scoring was performed as follows: 1-normal, 2-mild,3-moderate, 4-marked. These four scores were summed together andrepresented as an additional score, namely the ‘RA total score’. Toallow an inter-study comparison of the histology readouts, the overallhistology score of each rat was divided by the average overall histologyscore obtained for vehicle in the study to which that rat belongs andmultiplied by 100 (i.e. expressing the overall histology score as thepercentage of average vehicle overall histology score per study). Theaverage overall histology score of the different treatment groups wascalculated and compared.

3.1.10 Micro-Computed Tomography (μCT) Analysis of Calcaneus (Heel Bone)

Bone degradation observed in RA occurs especially at the cortical boneand can be revealed by μCT analysis (Sims N A et al., 2004; Oste L etal., ECTC Montreal 2007). After scanning and 3D volume reconstruction ofthe calcaneus bone, bone degradation was measured as the number ofdiscrete objects present per slide, isolated in silico perpendicular tothe longitudinal axis of the bone. The more the bone that was degraded,the more discrete objects that were measured. 1000 slices, evenlydistributed along the calcaneus (spaced by about 10.8 μm), are analyzed.

3.1.11 Results

Compound 1 was tested in the mouse CIA study at 30 mg/kg and in the ratCIA study at 30, 10, 3, and 1 mg/kg. Compound 1 was efficacious in allreadouts performed in the rat CIA study, with statistical significancein several of the readouts, in particular significant improvements wereseen for: the AUC of the clinical score (from 10 mg/kg), the endpointclinical score (from 1 mg/kg), the Larsen’ score (from 30 mg/kg) and pawswelling (from 1 mg/kg).

Example 3.2 Septic Shock Model

Injection of lipopolysaccharide (LPS) induces a rapid release of solubletumour necrosis factor (TNF-alpha) into the periphery. This model isused to analyse prospective blockers of TNF release in vivo.

Six BALB/cJ female mice (20 g) per group are treated at the intendeddosing once, po. Thirty minutes later, LPS (15 μg/kg; E. Coli serotype0111:B4) is injected ip. Ninety minutes later, mice are euthanized andblood is collected. Circulating TNF alpha levels are determined usingcommercially available ELISA kits. Dexamethasone (5 μg/kg) is used as areference anti-inflammatory compound. Selected compounds are tested atone or multiple doses, e.g. 3 and/or 10 and/or 30 mg/kg, po.

Compound 1 exhibited a statistically significant reduction in the TNFrelease (>50%) at 30 mg/kg po.

Example 3.3 MAB Model

The MAB model allows a rapid assessment of the modulation of an RA-likeinflammatory response by therapeutics (Kachigian L M. Nature Protocols(2006) 2512-2516: Collagen antibody-induced arthritis). DBA/J mice areinjected i.v. with a cocktail of mAbs directed against collagen II. Oneday later, compound treatment is initiated (vehicle: 10% (v/v) HPβCD).Three days later, mice receive an i.p. LPS injection (50 μg/mouse),resulting in a fast onset of inflammation. Compound treatment iscontinued until 10 days after the mAb injection. Inflammation is read bymeasuring paw swelling and recording the clinical score of each paw. Thecumulative clinical arthritis score of four limbs is presented to showthe severity of inflammation. A scoring system is applied to each limbusing a scale of 0-4, with 4 being the most severe inflammation.

-   -   0 Symptom free    -   1 Mild, but definite redness and swelling of one type of joint        such as the ankle or wrist, or apparent redness and swelling        limited to individual digits, regardless of the number of        affected digits    -   2 Moderate redness and swelling of two or more types of joints    -   3 Severe redness and swelling of the entire paw including digits    -   4 Maximally inflamed limb with involvement of multiple joints

Example 3.4 Oncology Models

In vivo models to validate efficacy of small molecules towardsJAK2-driven myeloproliferative diseases are described by Wernig et al.Cancer Cell 13, 311, 2008 and Geron et al. Cancer Cell 13, 321, 2008.

Example 3.5 Mouse IBD Model

In vitro and in vivo models to validate efficacy of small moleculestowards IBD are described by Wirtz et al. 2007.

Example 3.6 Mouse Asthma Model

In vitro and in vivo models to validate efficacy of small moleculestowards asthma are described by Nials et al., 2008; Ip et al. 2006;Pernis et al., 2002; Kudlacz et al., 2008.

Example 4 Toxicity, DMPK and Safety Models Example 4.1 ThermodynamicSolubility

A solution of 1 mg/mL of the test compound is prepared in a 0.2Mphosphate buffer pH7.4 or a 0.1M citrate buffer pH3.0 at roomtemperature in a glass vial.

The samples are rotated in a Rotator drive STR 4 (Stuart Scientific,Bibby) at speed 3.0 at room temperature for 24 hours.

After 24 hours, 800 μL of the sample is transferred to an eppendorf tubeand centrifuged 5 min at 14000 rpm. 200 μL of the supernatant of thesample is then transferred to a MultiscreenR Solubility Plate(Millipore, MSSLBPC50) and the supernatant is filtered (10-12″ Hg) withthe aid of a vacuum manifold into a clean Greiner polypropylene V-bottom96well plate (Cat no. 651201). 5 μL of the filtrate is diluted into 95μL (F20) of the same buffer used to incubate in the plate containing thestandard curve (Greiner, Cat no. 651201).

The standard curve for the compound is prepared freshly in DMSO startingfrom a 10 mM DMSO stock solution diluted factor 2 in DMSO (5000 μM) andthen further diluted in DMSO up to 19.5 μM. 3 μL of the dilution seriesas from 5000 μM is then transferred to a 97 μL acetonitrile-buffermixture (50/50). The final concentration range is 2.5 to 150 μM.

The plate is sealed with sealing mats (MA96RD-04S, www.kinesis.co.uk)and samples are measured at room temperature on LCMS (ZQ 1525 fromWaters) under optimized conditions using Quanoptimize to determine theappropriate mass of the molecule.

The samples are analyzed on LCMS with a flow rate of 1 mL/min. Solvent Ais 15 mM ammonia and solvent B is acetonitrile. The sample is run underpositive ion spray on an XBridge C18 3.5 μM (2.1×30 mm) column, fromWaters. The solvent gradient has a total run time of 2 minutes andranges from 5% B to 95% B.

Peak areas are analyzed with the aid of Masslynx software package andpeak areas of the samples are plotted against the standard curve toobtain the solubility of the compound.

Solubility values are reported in μM or μg/mL.

Example 4.2 Aqueous Solubility

Starting from a 10 mM stock in DMSO, a serial dilution of the compoundis prepared in DMSO. The dilution series is transferred to a 96 NUNCMaxisorb plate F-bottom (Cat no. 442404) and 0.2M phosphate buffer pH7.4 or 0.1M citrate buffer pH3.0 at room temperature is added.

The final concentration ranged from 200 μM to 2.5 μM in 5 equal dilutionsteps. The final DMSO concentration did not exceed 2%. 200 μM Pyrene isadded to the corner points of each 96 well plate and serves as areference point for calibration of Z-axis on the microscope.

The assay plates are sealed and incubated for 1 hour at 37° C. whileshaking at 230 rpm. The plates are then scanned under a white lightmicroscope, yielding individual pictures of the precipitate perconcentration. The precipitate is analyzed and converted into a numberwhich is plotted onto a graph. The first concentration at which thecompound appears completely dissolved is the concentration reported,however the true concentration lies somewhere between this concentrationand one dilution step higher.

Solubility values are reported in μg/mL

Example 4.3 Plasma Protein Binding (Equilibrium Dialysis)

A 10 mM stock solution of the compound in DMSO is diluted with a factor5 in DMSO. This solution is further diluted in freshly thawed human,rat, mouse or dog plasma (BioReclamation INC) with a final concentrationof 10 μM and final DMSO concentration of 0.5% (5.5 μl in 1094.5 μlplasma in a PP-Masterblock 96well (Greiner, Cat no. 780285))

A Pierce Red Device plate with inserts (ThermoScientific, Cat no. 89809)is prepared and filled with 750 μL PBS in the buffer chamber and 500 μLof the spiked plasma in the plasma chamber. The plate is incubated for 4hours at 37° C. while shaking at 230 rpm. After incubation, 120 μL ofboth chambers is transferred to 360 μL acetonitrile in a 96-well roundbottom, PP deep-well plates (Nunc, Cat no. 278743) and sealed with analuminum foil lid. The samples are mixed and placed on ice for 30 min.This plate is then centrifuged 30 min at 1200 rcf at 4° C. and thesupernatant is transferred to a 96 v-bottom PP plate (Greiner, 651201)for analysis on LCMS.

The plate is sealed with sealing mats (MA96RD-04S) of www.kinesis.co.ukand samples are measured at room temperature on LCMS (ZQ 1525 fromWaters) under optimized conditions using Quanoptimize to determine theappropriate mass of the molecule.

The samples are analyzed on LCMS with a flow rate of 1 mL/min. Solvent Awas 15 mM ammonia and solvent B was acetonitrile. The sample was rununder positive ion spray on an XBridge C18 3.5 μM (2.1×30 mm) column,from Waters. The solvent gradient has a total run time of 2 minutes andranges from 5% B to 95% B.

Peak area from the compound in the buffer chamber and the plasma chamberare considered to be 100% compound. The percentage bound to plasma isderived from these results and was reported to the LIMS as percentagebound to plasma.

The solubility of the compound in the final test concentration in PBS isinspected by microscope to indicate whether precipitation is observed ornot.

Example 4.4 Liability for QT Prolongation

Potential for QT prolongation is assessed in the hERG patch clamp assay.

4.4.1 Conventional Whole-Cell Patch-Clamp

Whole-cell patch-clamp recordings are performed using an EPC10 amplifiercontrolled by Pulse v8.77 software (HEKA). Series resistance istypically less than 10 MΩ and compensated by greater than 60%,recordings are not leak subtracted. Electrodes are manufactured fromGC150TF pipette glass (Harvard).

The external bathing solution contains: 135 mM NaCl, 5 mM KCl, 1.8 mMCaCl₂, 5 mM Glucose, 10 mM HEPES, pH 7.4.

The internal patch pipette solution contains: 100 mM Kgluconate, 20 mMKCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM Na₂ATP, 2 mM Glutathione, 11 mM EGTA,10 mM HEPES, pH 7.2.

Drugs are perfused using a Biologic MEV-9/EVH-9 rapid perfusion system.

All recordings are performed on HEK293 cells stably expressing hERGchannels. Cells are cultured on 12 mm round coverslips (German glass,Bellco) anchored in the recording chamber using two platinum rods(Goodfellow). hERG currents are evoked using an activating pulse to +40mV for 1000 ms followed by a tail current pulse to −50 mV for 2000 ms,holding potential was −80 mV. Pulses are applied every 20 s and allexperiments are performed at room temperature.

4.4.2 Data Analysis

IC₅₀ and IC₂₀ values are calculated for each compound tested. The folddifference between the IC₂₀ and the unbound C_(max) concentrations ofthe test compound obtained at relevant therapeutic doses as determinedby results obtained from the rat CIA model is calculated.

For the concentration response curves, peak tail current amplitude ismeasured during the voltage step to −50 mV. Curve-fitting ofconcentration-response data is performed using the equation:

y=a+[(b−a)/(1+10̂((log c−x)d)]

where a is minimum response, b is maximum response and d is Hill slope,this equation can be used to calculate both IC₅₀ (where y=50 and c isthe IC₅₀ value) and IC₂₀ (where y=20 and c is the IC₂₀ value). GraphPad®Prism® (Graphpad® Software Inc.) software was used for all curvefitting. A difference of 100 fold or greater indicates a low potentialfor QT prolongation.

Example 4.5 Microsomal Stability

A 10 mM stock solution of compound in DMSO was diluted 1000 fold in a182 mM phosphate buffer pH7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-incubated at 37° C.

40 μL of deionised water was added to a well of a polypropylene Matrix2D barcode labelled storage tube (Thermo Scientific) and pre-incubatedat 37° C.

A Glucose-6-phophate-dehydrogenase (G6PDH) working stock solution wasprepared in 182 mM phosphate buffer pH7.4 and placed on ice before use.A co-factor containing MgCl₂, glucose-6-phosphate and NADP+ was preparedin deionised water and placed on ice before use.

A final working solution containing liver microsomes (Xenotech) of aspecies of interest (human, mouse, rat, dog), previously described G6PDHand co-factors was prepared and this mix was incubated for no longerthan 20 minutes at room temperature.

30 μL of the pre-heated compound dilution was added to 40 μL ofpre-heated water in the Matrix tubes and 30 μL of the microsomal mix wasadded. Final reaction concentrations were 3 μM compound, 1 mgmicrosomes, 0.4 U/mL GDPDH, 3.3 mM MgCl₂, 3.3 mM glucose-6-phosphate and1.3 mM NADP+.

To measure percentage remaining of compound at time zero MeOH or ACN wasadded (1:1) to the well before adding the microsomal mix. The plateswere sealed with Matrix Sepra Seals™ (Matrix, Cat. No. 4464) and shakenfor a few seconds ensure complete mixing of all components.

The samples which were not stopped are incubated at 37° C., 300 rpm andafter 1 hour of incubation the reaction was stopped with MeOH or ACN(1:1).

After stopping the reaction the samples were mixed and placed on ice for30 min to precipitate the proteins. The plates were then centrifuged 30min at 1200 rcf at 4° C. and the supernatant was transferred to a 96v-bottom PP plate (Greiner, 651201) for analysis on LCMS.

These plates were sealed with sealing mats (MA96RD-04S) ofwww.kinesis.co.uk and samples were measured at room temperature on LCMS(ZQ 1525 from Waters) under optimized conditions using Quanoptimize todetermine the appropriate mass of the parent molecule.

The samples were analyzed on LCMS with a flow rate of 1 mL/min. SolventA was 15 mM ammonia and solvent B was methanol or acetonitrile,depending on the stop solution used. The samples were run under positiveion spray on an XBridge C18 3.5 μM (2.1×30 mm) column, from Waters. Thesolvent gradient had a total run time of 2 minutes and ranges from 5% Bto 95% B.

Peak area from the parent compound at time 0 was considered to be 100%remaining. The percentage remaining after 1 hour incubation wascalculated from time 0 and was calculated as the percentage remaining.The solubility of the compound in the final test concentration in bufferis inspected by microscope and results are reported.

The data on microsomal stability are expressed as a percentage of thetotal amount of compound remaining after 60 minutes.

TABLE IX Microsomal stability Compound # Human Rat 1 76-100% 76-100%

Example 4.6 Caco2 Permeability

Bi-directional Caco-2 assays were performed as described below. Caco-2cells were obtained from European Collection of Cell Cultures (ECACC,cat 86010202) and used after a 21 day cell culture in 24-well Transwellplates (Fisher TKT-545-020B).

2×10⁵ cells/well were seeded in plating medium consisting ofDMEM+GlutaMAXI+1% NEAA+10% FBS (FetalClone II)+1% Pen/Strep. The mediumwas changed every 2-3 days.

Test and reference compounds (propranolol and rhodamine-123 orvinblastine, all purchased from Sigma) were prepared in Hanks' BalancedSalt Solution containing 25 mM HEPES (pH7.4) and added to either theapical (125 μL) or basolateral (600 μL) chambers of the Transwell plateassembly at a concentration of 10 μM with a final DMSO concentration of0.25%.

50 μM Lucifer Yellow (Sigma) was added to the donor buffer in all wellsto assess integrity of the cell layers by monitoring Lucifer Yellowpermeation. As Lucifer Yellow (LY) cannot freely permeate lipophilicbarriers, a high degree of LY transport indicates poor integrity of thecell layer.

After a 1 hour incubation at 37° C. while shaking at an orbital shakerat 150 rpm, 70 μL aliquots were taken from both apical (A) and basal (B)chambers and added to 100 μl 50:50 acetonitrile:water solutioncontaining analytical internal standard (0.5 μM carbamazepine) in a 96well plate.

Lucifer yellow was measured with a Spectramax Gemini XS (Ex 426 nm andEm 538 nm) in a clean 96 well plate containing 150 μL of liquid frombasolateral and apical side.

Concentrations of compound in the samples were measured by highperformance liquid-chromatography/mass spectroscopy (LC-MS/MS).

Apparent permeability (P_(app)) values were calculated from therelationship:

P _(app)=[compound]_(acceptor final) ×V_(acceptor)/([compound]_(donor initial) ×V _(donor))/T _(inc) ×V_(donor)/surface area×60×10⁻⁶ cm/s

V=chamber volumeT_(ine)=incubation time.Surface area=0.33 cm²

The Efflux ratios, as an indication of active efflux from the apicalcell surface, were calculated using the ratio of P_(app) B>A/P_(app)A>B.

The following assay acceptance criteria were used:

Propranolol: P_(app) (A>B) value ≧20(×10⁻⁶ cm/s)Rhodamine 123 or Vinblastine: P_(app) (A>B) value <5 (×10⁻⁶ cm/s) withEfflux ratio ≧5.Lucifer yellow permeability: ≦100 nm/s

TABLE X Caco2 Efflux rate Papp A > B Compound # (×10−6 cm/sec) Effluxratio 1 7.8 ± 0.8 6.1 ± 0.6

Example 4.7 Pharmacokinetic Study 4.7.1 Pharmacokinetic Study in Rodents

Compounds are formulated in PEG200/physiological saline orPEG400/DMSO/physiological saline mixtures for the intravenous route andin 0.5% methylcellulose or 10-30% hydroxylpropyl-β-cyclodextrine pH3 orpH7.4 for the oral route. Test compounds are orally dosed as a singleesophageal gavage at 5-10 mg/kg and intravenously dosed as a bolus viathe caudal vein at 1 mg/kg. Each group consists of 3 rats. Blood samplesare collected either via the jugular vein using cannulated rats or atthe retro-orbital sinus with lithium heparin as anti-coagulant at thetime points in the following range: 0.05 to 8 hours (intravenous route),and 0.25 to 6 or 24 hours (oral route). Whole blood samples arecentrifuged at 5000 rpm for 10 min and the resulting plasma samples arestored at −20° C. pending analysis.

4.7.2 Pharmacokinetic Study in Dogs

Compounds are formulated in PEG200/physiological saline mixtures for theintravenous route and in 0.5% methylcellulose orPEG400/hydroxylpropyl-β-cyclodextrine mixtures acidified with citricacid to pH2-3 for the oral route. Test compounds are orally dosed viagavage at 5-30 mg/kg and intravenously dosed as a bolus or a 10 mininfusion via the cephalic vein at 1 mg/kg. Each group consists of 3 maleor female Beagle dogs. Blood samples are collected via the jugular veinwith lithium heparin as anti-coagulant at time points between 0.083 to24 hours postdose. Whole blood samples are centrifuged at 5000 rpm for10 min and the resulting plasma samples are stored at −20° C. pendinganalysis.

4.7.3 Quantification of Compound Levels in Plasma

Plasma concentrations of each test compound are determined by anLC-MS/MS method in which the mass spectrometer is operated in positiveelectrospray mode.

4.7.4 Determination of Pharmacokinetic Parameters

Pharmacokinetic parameters are calculated using Winnonlin® (Pharsight®,United).

Example 4.8 7-Day Rat Toxicity Study

A 7-day oral toxicity study with test compounds is performed inSprague-Dawley male rats to assess their toxic potential andtoxicokinetics, at daily doses of 100, 300 and 500 mg/kg/day, by gavage,at the constant dosage-volume of 5 mL/kg/day.

The test compounds are formulated in 30% (v/v) HPβCD in purified water.Each group includes 5 principal male rats as well as 3 satellite animalsfor toxicokinetics. A fourth group is given 30% (v/v) HPβCD in wateronly, at the same frequency, dosage volume and by the same route ofadministration, and acts as the vehicle control group.

The goal of the study is to determine the lowest dose that results in noadverse events being identified (no observable adverse effectlevel—NOAEL).

Example 4.9 Hepatocyte Stability

Models to evaluate metabolic clearance in hepatocyte are described byMcGinnity et al. Drug Metabolism and Disposition 2008, 32, 11, 1247.

It will be appreciated by those skilled in the art that the foregoingdescriptions are exemplary and explanatory in nature, and as indicatedare intended to illustrate the invention and its preferred embodiments.Through routine experimentation, an artisan will recognise apparentmodifications and variations that may be made without departing from thespirit of the invention. Thus, the invention is intended to be definednot by the above description, but by the following claims and theirequivalents.

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All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

From the foregoing description, various modifications and changes in thecompositions and methods of this invention will occur to those skilledin the art. All such modifications coming within the scope of theappended claims are intended to be included therein.

It should be understood that factors such as the differential cellpenetration capacity of the various compounds can contribute todiscrepancies between the activity of the compounds in the in vitrobiochemical and cellular assays.

At least some of the chemical names of compounds of the invention asgiven and set forth in this application, may have been generated on anautomated basis by use of a commercially available chemical namingsoftware program, and have not been independently verified.Representative programs performing this function include the Lexichemnaming tool sold by Open Eye Software, Inc. and the Autonom Softwaretool sold by MDL, Inc. In the instance where the indicated chemical nameand the depicted structure differ, the depicted structure will control.

Chemical structures shown herein were prepared using either ChemDraw® orISIS®/DRAW. Any open valency appearing on a carbon, oxygen or nitrogenatom in the structures herein indicates the presence of a hydrogen atom.Where a chiral center exists in a structure but no specificstereochemistry is shown for the chiral center, both enantiomersassociated with the chiral structure are encompassed by the structure.

1. A compound according to Formula I:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and apharmaceutically effective amount of a compound, or pharmaceuticallyacceptable salt thereof, according to claim
 1. 3. The pharmaceuticalcomposition according to claim 2 comprising a further therapeutic agent.4. (canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. A method forthe treatment or prophylaxis of inflammatory conditions, autoimmunediseases, proliferative diseases, transplantation rejection, diseasesinvolving impairment of cartilage turnover, congenital cartilagemalformations, and/or diseases associated with hypersecretion of IL6,said method comprising administering an effective amount of a compound,or pharmaceutically acceptable salt thereof, according to claim
 1. 9. Amethod according to claim 8, wherein the inflammatory condition isrheumatoid arthritis.
 10. A method according to claim 8, wherein thecondition or disease involves inflammation.
 11. The method according toclaim 8, wherein the compound according to claim 1 is administered incombination with a further therapeutic agent.
 12. The pharmaceuticalcomposition according to claim 2, wherein the further therapeutic agentis an agent for the treatment, prevention or prophylaxis of inflammatoryconditions, autoimmune diseases, proliferative diseases, transplantationrejection, diseases involving impairment of cartilage turnover,congenital cartilage malformations, and/or diseases associated withhypersecretion of IL6.
 13. A method for the treatment or prophylaxis ofinflammatory conditions, autoimmune diseases, proliferative diseases,transplantation rejection, diseases involving impairment of cartilageturnover, congenital cartilage malformations, and/or diseases associatedwith hypersecretion of IL6, said method comprising administering aneffective amount of a pharmaceutical composition according to claim 2.14. A method according to claim 13, wherein the inflammatory conditionis rheumatoid arthritis.
 15. A method according to claim 13, wherein thecondition or disease involves inflammation.
 16. The method according toclaim 13, wherein the compound according to claim 1 is administered incombination with a further therapeutic agent.
 17. The method accordingto claim 11, wherein the further therapeutic agent is an agent for thetreatment, prevention or prophylaxis of inflammatory conditions,autoimmune diseases, proliferative diseases, transplantation rejection,diseases involving impairment of cartilage turnover, congenitalcartilage malformations, and/or diseases associated with hypersecretionof IL6.
 18. The method according to claim 16, wherein the furthertherapeutic agent is an agent for the treatment, prevention orprophylaxis of inflammatory conditions, autoimmune diseases,proliferative diseases, transplantation rejection, diseases involvingimpairment of cartilage turnover, congenital cartilage malformations,and/or diseases associated with hypersecretion of IL6.